CRISPR-Cas systems and methods for altering expression of gene products

ABSTRACT

The invention provides for systems, methods, and compositions for altering expression of target gene sequences and related gene products. Provided are vectors and vector systems, some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for utilizing the CRISPR-Cas system.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a continuation of U.S. patent application Ser. No.14/054,414 filed Oct. 15, 2013 which claims priority to U.S. provisionalpatent application 61/842,322 having Broad reference BI-2011/008A,entitled CRISPR-CAS SYSTEMS AND METHODS FOR ALTERING EXPRESSION OF GENEPRODUCTS filed on Jul. 2, 2013. Priority is also claimed to U.S.provisional patent applications 61/736,527, 61/748,427, 61/791,409 and61/835,931 having Broad reference BI-2011/008/WSGR, BI-2011/008/WSGR,Broad reference BI-2011/008/VP and BI-2011/008/VP respectively, allentitled SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATIONfiled on Dec. 12, 2012, Jan. 2, 2013, Mar. 15, 2013 and Jun. 17, 2013,respectively.

The foregoing applications, and all documents cited therein or duringtheir prosecution (“appln cited documents”) and all documents cited orreferenced in the appln cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under NIH Pioneer AwardDP1MH100706, awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention generally relates to systems, methods andcompositions used for the control of gene expression involving sequencetargeting, such as genome perturbation or gene-editing, that may usevector systems related to Clustered Regularly Interspaced ShortPalindromic Repeats (CRISPR) and components thereof.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 6, 2013, isnamed 44790.05.2003_SL.txt and is 56,781 bytes in size.

BACKGROUND OF THE INVENTION

Recent advances in genome sequencing techniques and analysis methodshave significantly accelerated the ability to catalog and map geneticfactors associated with a diverse range of biological functions anddiseases. Precise genome targeting technologies are needed to enablesystematic reverse engineering of causal genetic variations by allowingselective perturbation of individual genetic elements, as well as toadvance synthetic biology, biotechnological, and medical applications.Although genome-editing techniques such as designer zinc fingers,transcription activator-like effectors (TALEs), or homing meganucleasesare available for producing targeted genome perturbations, there remainsa need for new genome engineering technologies that are affordable, easyto set up, scalable, and amenable to targeting multiple positions withinthe eukaryotic genome.

SUMMARY OF THE INVENTION

There exists a pressing need for alternative and robust systems andtechniques for sequence targeting with a wide array of applications.This invention addresses this need and provides related advantages. TheCRISPR/Cas or the CRISPR-Cas system (both terms are used interchangeablythroughout this application) does not require the generation ofcustomized proteins to target specific sequences but rather a single Casenzyme can be programmed by a short RNA molecule to recognize a specificDNA target, in other words the Cas enzyme can be recruited to a specificDNA target using said short RNA molecule. Adding the CRISPR-Cas systemto the repertoire of genome sequencing techniques and analysis methodsmay significantly simplify the methodology and accelerate the ability tocatalog and map genetic factors associated with a diverse range ofbiological functions and diseases. To utilize the CRISPR-Cas systemeffectively for genome editing without deleterious effects, it iscritical to understand aspects of engineering and optimization of thesegenome engineering tools, which are aspects of the claimed invention.

In one aspect, the invention provides a method for altering or modifyingexpression of one or more gene products. The said method may compriseintroducing into a eukaryotic cell containing and expressing DNAmolecules encoding the one or more gene products an engineered,non-naturally occurring vector system comprising one or more vectorscomprising: a) a first regulatory element operably linked to one or moreClustered Regularly Interspaced Short Palindromic Repeats(CRISPR)—CRISPR associated (Cas) system guide RNAs that hybridize withtarget sequences in genomic loci of the DNA molecules encoding the oneor more gene products, b) a second regulatory element operably linked toa Type-II Cas9 protein, wherein components (a) and (b) are located onsame or different vectors of the system, whereby the guide RNAs targetthe genomic loci of the DNA molecules encoding the one or more geneproducts and the Cas9 protein cleaves the genomic loci of the DNAmolecules encoding the one or more gene products, whereby expression ofthe one or more gene products is altered; and, wherein the Cas9 proteinand the guide RNAs do not naturally occur together. The inventioncomprehends the expression of two or more gene products being alteredand the vectors of the system further comprising one or more nuclearlocalization signal(s) (NLS(s)). The invention comprehends the guideRNAs comprising a guide sequence fused to a tracr sequence. Theinvention further comprehends the Cas9 protein being codon optimized forexpression in the eukaryotic cell. In a preferred embodiment theeukaryotic cell is a mammalian cell or a human cell. In a furtherembodiment of the invention, the expression of one or more of the geneproducts is decreased. In aspects of the invention cleaving the genomicloci of the DNA molecule encoding the gene product encompasses cleavingeither one or both strands of the DNA duplex.

In one aspect, the invention provides an engineered, programmable,non-naturally occurring CRISPR-Cas system comprising a Cas9 protein andone or more guide RNAs that target the genomic loci of DNA moleculesencoding one or more gene products in a eukaryotic cell and the Cas9protein cleaves the genomic loci of the DNA molecules encoding the oneor more gene products, whereby expression of the one or more geneproducts is altered; and, wherein the Cas9 protein and the guide RNAs donot naturally occur together. The invention comprehends the expressionof two or more gene products being altered and the CRISPR-Cas systemfurther comprising one or more NLS(s). The invention comprehends theguide RNAs comprising a guide sequence fused to a tracr sequence. Theinvention further comprehends the Cas9 protein being codon optimized forexpression in the eukaryotic cell. In a preferred embodiment theeukaryotic cell is a mammalian cell or a human cell. In aspects of theinvention cleaving the genomic loci of the DNA molecule encoding thegene product encompasses cleaving either one or both strands of the DNAduplex.

In another aspect, the invention provides an engineered, non-naturallyoccurring vector system comprising one or more vectors comprising a) afirst regulatory element operably linked to one or more CRISPR-Cassystem guide RNAs that hybridize with target sequences in genomic lociof DNA molecules encoding one or more gene products, b) a secondregulatory element operably linked to a Type-II Cas9 protein, whereincomponents (a) and (b) are located on same or different vectors of thesystem, whereby the guide RNAs target the genomic loci of the DNAmolecules encoding the one or more gene products in a eukaryotic celland the Cas9 protein cleaves the genomic loci of the DNA moleculesencoding the one or more gene products, whereby expression of the one ormore gene products is altered; and, wherein the Cas9 protein and theguide RNAs do not naturally occur together. The invention comprehendsthe expression of two or more gene products being altered and thevectors of the system further comprising one or more nuclearlocalization signal(s) (NLS(s)). The invention comprehends the guideRNAs comprising a guide sequence fused to a tracr sequence. Theinvention further comprehends the Cas9 protein being codon optimized forexpression in the eukaryotic cell. In a preferred embodiment theeukaryotic cell is a mammalian cell or a human cell. In a furtherembodiment of the invention, the expression of one or more of the geneproducts is decreased. In aspects of the invention cleaving the genomicloci of the DNA molecule encoding the gene product encompasses cleavingeither one or both strands of the DNA duplex.

In one aspect, the invention provides a vector system comprising one ormore vectors. In some embodiments, the system comprises: (a) a firstregulatory element operably linked to a tracr mate sequence and one ormore insertion sites for inserting one or more guide sequences upstreamof the tracr mate sequence, wherein when expressed, the guide sequencedirects sequence-specific binding of a CRISPR complex to a targetsequence in a eukaryotic cell, wherein the CRISPR complex comprises aCRISPR enzyme complexed with (1) the guide sequence that is hybridizedto the target sequence, and (2) the tracr mate sequence that ishybridized to the tracr sequence; and (b) a second regulatory elementoperably linked to an enzyme-coding sequence encoding said CRISPR enzymecomprising a nuclear localization sequence; wherein components (a) and(b) are located on the same or different vectors of the system. In someembodiments, component (a) further comprises the tracr sequencedownstream of the tracr mate sequence under the control of the firstregulatory element. In some embodiments, component (a) further comprisestwo or more guide sequences operably linked to the first regulatoryelement, wherein when expressed, each of the two or more guide sequencesdirect sequence specific binding of a CRISPR complex to a differenttarget sequence in a eukaryotic cell. In some embodiments, the systemcomprises the tracr sequence under the control of a third regulatoryelement, such as a polymerase III promoter. In some embodiments, thetracr sequence exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% ofsequence complementarity along the length of the tracr mate sequencewhen optimally aligned. Determining optimal alignment is within thepurview of one of skill in the art. For example, there are publicallyand commercially available alignment algorithms and programs such as,but not limited to, ClustalW, Smith-Waterman in matlab, Bowtie,Geneious, Biopython and SeqMan. In some embodiments, the CRISPR complexcomprises one or more nuclear localization sequences of sufficientstrength to drive accumulation of said CRISPR complex in a detectableamount in the nucleus of a eukaryotic cell. Without wishing to be boundby theory, it is believed that a nuclear localization sequence is notnecessary for CRISPR complex activity in eukaryotes, but that includingsuch sequences enhances activity of the system, especially as totargeting nucleic acid molecules in the nucleus. In some embodiments,the CRISPR enzyme is a type II CRISPR system enzyme. In someembodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments,the Cas9 enzyme is S. pneumoniae, S. pyogenes, or S. thermophilus Cas9,and may include mutated Cas9 derived from these organisms. The enzymemay be a Cas9 homolog or ortholog. In some embodiments, the CRISPRenzyme is codon-optimized for expression in a eukaryotic cell. In someembodiments, the CRISPR enzyme directs cleavage of one or two strands atthe location of the target sequence. In some embodiments, the firstregulatory element is a polymerase III promoter. In some embodiments,the second regulatory element is a polymerase II promoter. In someembodiments, the guide sequence is at least 15, 16, 17, 18, 19, 20, 25nucleotides, or between 10-30, or between 15-25, or between 15-20nucleotides in length. In general, and throughout this specification,the term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. Vectorsinclude, but are not limited to, nucleic acid molecules that aresingle-stranded, double-stranded, or partially double-stranded; nucleicacid molecules that comprise one or more free ends, no free ends (e.g.circular); nucleic acid molecules that comprise DNA, RNA, or both; andother varieties of polynucleotides known in the art. One type of vectoris a “plasmid,” which refers to a circular double stranded DNA loop intowhich additional DNA segments can be inserted, such as by standardmolecular cloning techniques. Another type of vector is a viral vector,wherein virally-derived DNA or RNA sequences are present in the vectorfor packaging into a virus (e.g. retroviruses, replication defectiveretroviruses, adenoviruses, replication defective adenoviruses, andadeno-associated viruses). Viral vectors also include polynucleotidescarried by a virus for transfection into a host cell. Certain vectorsare capable of autonomous replication in a host cell into which they areintroduced (e.g. bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively-linked.Such vectors are referred to herein as “expression vectors.” Commonexpression vectors of utility in recombinant DNA techniques are often inthe form of plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell).

The term “regulatory element” is intended to include promoters,enhancers, internal ribosomal entry sites (IRES), and other expressioncontrol elements (e.g. transcription termination signals, such aspolyadenylation signals and poly-U sequences). Such regulatory elementsare described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).Regulatory elements include those that direct constitutive expression ofa nucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). A tissue-specific promoter maydirect expression primarily in a desired tissue of interest, such asmuscle, neuron, bone, skin, blood, specific organs (e.g. liver,pancreas), or particular cell types (e.g. lymphocytes). Regulatoryelements may also direct expression in a temporal-dependent manner, suchas in a cell-cycle dependent or developmental stage-dependent manner,which may or may not also be tissue or cell-type specific. In someembodiments, a vector comprises one or more pol III promoter (e.g. 1, 2,3, 4, 5, or more pol I promoters), one or more pol II promoters (e.g. 1,2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g.1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof.Examples of pol II promoters include, but are not limited to, U6 and H1promoters. Examples of pol II promoters include, but are not limited to,the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally withthe RSV enhancer), the cytomegalovirus (CMV) promoter (optionally withthe CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)],the SV40 promoter, the dihydrofolate reductase promoter, the β-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the EF1αpromoter. Also encompassed by the term “regulatory element” are enhancerelements, such as WPRE; CMV enhancers; the R-U5′ segment in LTR ofHTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer;and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc.Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). It will beappreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression desired, etc. A vectorcan be introduced into host cells to thereby produce transcripts,proteins, or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., clustered regularlyinterspersed short palindromic repeats (CRISPR) transcripts, proteins,enzymes, mutant forms thereof, fusion proteins thereof, etc.).

Advantageous vectors include lentiviruses and adeno-associated viruses,and types of such vectors can also be selected for targeting particulartypes of cells.

In one aspect, the invention provides a eukaryotic host cell comprising(a) a first regulatory element operably linked to a tracr mate sequenceand one or more insertion sites for inserting one or more guidesequences upstream of the tracr mate sequence, wherein when expressed,the guide sequence directs sequence-specific binding of a CRISPR complexto a target sequence in a eukaryotic cell, wherein the CRISPR complexcomprises a CRISPR enzyme complexed with (1) the guide sequence that ishybridized to the target sequence, and (2) the tracr mate sequence thatis hybridized to the tracr sequence; and/or (b) a second regulatoryelement operably linked to an enzyme-coding sequence encoding saidCRISPR enzyme comprising a nuclear localization sequence. In someembodiments, the host cell comprises components (a) and (b). In someembodiments, component (a), component (b), or components (a) and (b) arestably integrated into a genome of the host eukaryotic cell. In someembodiments, component (a) further comprises the tracr sequencedownstream of the tracr mate sequence under the control of the firstregulatory element. In some embodiments, component (a) further comprisestwo or more guide sequences operably linked to the first regulatoryelement, wherein when expressed, each of the two or more guide sequencesdirect sequence specific binding of a CRISPR complex to a differenttarget sequence in a eukaryotic cell. In some embodiments, theeukaryotic host cell further comprises a third regulatory element, suchas a polymerase III promoter, operably linked to said tracr sequence. Insome embodiments, the tracr sequence exhibits at least 50%, 60%, 70%,80%, 90%, 95%, or 99% of sequence complementarity along the length ofthe tracr mate sequence when optimally aligned. The enzyme may be a Cas9homolog or ortholog. In some embodiments, the CRISPR enzyme iscodon-optimized for expression in a eukaryotic cell. In someembodiments, the CRISPR enzyme directs cleavage of one or two strands atthe location of the target sequence. In some embodiments, the CRISPRenzyme lacks DNA strand cleavage activity. In some embodiments, thefirst regulatory element is a polymerase III promoter. In someembodiments, the second regulatory element is a polymerase II promoter.In some embodiments, the guide sequence is at least 15, 16, 17, 18, 19,20, 25 nucleotides, or between 10-30, or between 15-25, or between 15-20nucleotides in length. In an aspect, the invention provides a non-humaneukaryotic organism; preferably a multicellular eukaryotic organism,comprising a eukaryotic host cell according to any of the describedembodiments. In other aspects, the invention provides a eukaryoticorganism; preferably a multicellular eukaryotic organism, comprising aeukaryotic host cell according to any of the described embodiments. Theorganism in some embodiments of these aspects may be an animal; forexample a mammal. Also, the organism may be an arthropod such as aninsect. The organism also may be a plant. Further, the organism may be afungus.

In one aspect, the invention provides a kit comprising one or more ofthe components described herein. In some embodiments, the kit comprisesa vector system and instructions for using the kit. In some embodiments,the vector system comprises (a) a first regulatory element operablylinked to a tracr mate sequence and one or more insertion sites forinserting one or more guide sequences upstream of the tracr matesequence, wherein when expressed, the guide sequence directssequence-specific binding of a CRISPR complex to a target sequence in aeukaryotic cell, wherein the CRISPR complex comprises a CRISPR enzymecomplexed with (1) the guide sequence that is hybridized to the targetsequence, and (2) the tracr mate sequence that is hybridized to thetracr sequence; and/or (b) a second regulatory element operably linkedto an enzyme-coding sequence encoding said CRISPR enzyme comprising anuclear localization sequence. In some embodiments, the kit comprisescomponents (a) and (b) located on the same or different vectors of thesystem. In some embodiments, component (a) further comprises the tracrsequence downstream of the tracr mate sequence under the control of thefirst regulatory element. In some embodiments, component (a) furthercomprises two or more guide sequences operably linked to the firstregulatory element, wherein when expressed, each of the two or moreguide sequences direct sequence specific binding of a CRISPR complex toa different target sequence in a eukaryotic cell. In some embodiments,the system further comprises a third regulatory element, such as apolymerase III promoter, operably linked to said tracr sequence. In someembodiments, the tracr sequence exhibits at least 50%, 60%, 70%, 80%,90%, 95%, or 99% of sequence complementarity along the length of thetracr mate sequence when optimally aligned. In some embodiments, theCRISPR enzyme comprises one or more nuclear localization sequences ofsufficient strength to drive accumulation of said CRISPR enzyme in adetectable amount in the nucleus of a eukaryotic cell. In someembodiments, the CRISPR enzyme is a type II CRISPR system enzyme. Insome embodiments, the CRISPR enzyme is a Cas9 enzyme. In someembodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes or S.thermophilus Cas9, and may include mutated Cas9 derived from theseorganisms. The enzyme may be a Cas9 homolog or ortholog. In someembodiments, the CRISPR enzyme is codon-optimized for expression in aeukaryotic cell. In some embodiments, the CRISPR enzyme directs cleavageof one or two strands at the location of the target sequence. In someembodiments, the CRISPR enzyme lacks DNA strand cleavage activity. Insome embodiments, the first regulatory element is a polymerase IIIpromoter. In some embodiments, the second regulatory element is apolymerase II promoter. In some embodiments, the guide sequence is atleast 15, 16, 17, 18, 19, 20, 25 nucleotides, or between 10-30, orbetween 15-25, or between 15-20 nucleotides in length.

In one aspect, the invention provides a method of modifying a targetpolynucleotide in a eukaryotic cell. In some embodiments, the methodcomprises allowing a CRISPR complex to bind to the target polynucleotideto effect cleavage of said target polynucleotide thereby modifying thetarget polynucleotide, wherein the CRISPR complex comprises a CRISPRenzyme complexed with a guide sequence hybridized to a target sequencewithin said target polynucleotide, wherein said guide sequence is linkedto a tracr mate sequence which in turn hybridizes to a tracr sequence.In some embodiments, said cleavage comprises cleaving one or two strandsat the location of the target sequence by said CRISPR enzyme. In someembodiments, said cleavage results in decreased transcription of atarget gene. In some embodiments, the method further comprises repairingsaid cleaved target polynucleotide by homologous recombination with anexogenous template polynucleotide, wherein said repair results in amutation comprising an insertion, deletion, or substitution of one ormore nucleotides of said target polynucleotide. In some embodiments,said mutation results in one or more amino acid changes in a proteinexpressed from a gene comprising the target sequence. In someembodiments, the method further comprises delivering one or more vectorsto said eukaryotic cell, wherein the one or more vectors driveexpression of one or more of: the CRISPR enzyme, the guide sequencelinked to the tracr mate sequence, and the tracr sequence. In someembodiments, said vectors are delivered to the eukaryotic cell in asubject. In some embodiments, said modifying takes place in saideukaryotic cell in a cell culture. In some embodiments, the methodfurther comprises isolating said eukaryotic cell from a subject prior tosaid modifying. In some embodiments, the method further comprisesreturning said eukaryotic cell and/or cells derived therefrom to saidsubject.

In one aspect, the invention provides a method of modifying expressionof a polynucleotide in a eukaryotic cell. In some embodiments, themethod comprises allowing a CRISPR complex to bind to the polynucleotidesuch that said binding results in increased or decreased expression ofsaid polynucleotide; wherein the CRISPR complex comprises a CRISPRenzyme complexed with a guide sequence hybridized to a target sequencewithin said polynucleotide, wherein said guide sequence is linked to atracr mate sequence which in turn hybridizes to a tracr sequence. Insome embodiments, the method further comprises delivering one or morevectors to said eukaryotic cells, wherein the one or more vectors driveexpression of one or more of: the CRISPR enzyme, the guide sequencelinked to the tracr mate sequence, and the tracr sequence.

In one aspect, the invention provides a method of generating a modeleukaryotic cell comprising a mutated disease gene. In some embodiments,a disease gene is any gene associated an increase in the risk of havingor developing a disease. In some embodiments, the method comprises (a)introducing one or more vectors into a eukaryotic cell, wherein the oneor more vectors drive expression of one or more of: a CRISPR enzyme, aguide sequence linked to a tracr mate sequence, and a tracr sequence;and (b) allowing a CRISPR complex to bind to a target polynucleotide toeffect cleavage of the target polynucleotide within said disease gene,wherein the CRISPR complex comprises the CRISPR enzyme complexed with(1) the guide sequence that is hybridized to the target sequence withinthe target polynucleotide, and (2) the tracr mate sequence that ishybridized to the tracr sequence, thereby generating a model eukaryoticcell comprising a mutated disease gene. In some embodiments, saidcleavage comprises cleaving one or two strands at the location of thetarget sequence by said CRISPR enzyme. In some embodiments, saidcleavage results in decreased transcription of a target gene. In someembodiments, the method further comprises repairing said cleaved targetpolynucleotide by homologous recombination with an exogenous templatepolynucleotide, wherein said repair results in a mutation comprising aninsertion, deletion, or substitution of one or more nucleotides of saidtarget polynucleotide. In some embodiments, said mutation results in oneor more amino acid changes in a protein expression from a genecomprising the target sequence.

In one aspect, the invention provides a method for developing abiologically active agent that modulates a cell signaling eventassociated with a disease gene. In some embodiments, a disease gene isany gene associated an increase in the risk of having or developing adisease. In some embodiments, the method comprises (a) contacting a testcompound with a model cell of any one of the described embodiments; and(b) detecting a change in a readout that is indicative of a reduction oran augmentation of a cell signaling event associated with said mutationin said disease gene, thereby developing said biologically active agentthat modulates said cell signaling event associated with said diseasegene.

In one aspect, the invention provides a recombinant polynucleotidecomprising a guide sequence upstream of a tracr mate sequence, whereinthe guide sequence when expressed directs sequence-specific binding of aCRISPR complex to a corresponding target sequence present in aeukaryotic cell. In some embodiments, the target sequence is a viralsequence present in a eukaryotic cell. In some embodiments, the targetsequence is a proto-oncogene or an oncogene.

In one aspect the invention provides for a method of selecting one ormore cell(s) by introducing one or more mutations in a gene in the oneor more cell (s), the method comprising: introducing one or more vectorsinto the cell (s), wherein the one or more vectors drive expression ofone or more of: a CRISPR enzyme, a guide sequence linked to a tracr matesequence, a tracr sequence, and an editing template; wherein the editingtemplate comprises the one or more mutations that abolish CRISPR enzymecleavage; allowing homologous recombination of the editing template withthe target polynucleotide in the cell(s) to be selected; allowing aCRISPR complex to bind to a target polynucleotide to effect cleavage ofthe target polynucleotide within said gene, wherein the CRISPR complexcomprises the CRISPR enzyme complexed with (1) the guide sequence thatis hybridized to the target sequence within the target polynucleotide,and (2) the tracr mate sequence that is hybridized to the tracrsequence, wherein binding of the CRISPR complex to the targetpolynucleotide induces cell death, thereby allowing one or more cell(s)in which one or more mutations have been introduced to be selected. In apreferred embodiment, the CRISPR enzyme is Cas9. In another preferredembodiment of the invention the cell to be selected may be a eukaryoticcell. Aspects of the invention allow for selection of specific cellswithout requiring a selection marker or a two-step process that mayinclude a counter-selection system Accordingly, it is an object of theinvention not to encompass within the invention any previously knownproduct, process of making the product, or method of using the productsuch that Applicants reserve the right and hereby disclose a disclaimerof any previously known product, process, or method. It is further notedthat the invention does not intend to encompass within the scope of theinvention any product, process, or making of the product or method ofusing the product, which does not meet the written description andenablement requirements of the USPTO (35 U.S.C. §112, first paragraph)or the EPO (Article 83 of the EPC), such that Applicants reserve theright and hereby disclose a disclaimer of any previously describedproduct, process of making the product, or method of using the product.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention. These and other embodiments aredisclosed or are obvious from and encompassed by, the following DetailedDescription.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 shows a schematic model of the CRISPR system. The Cas9 nucleasefrom Streptococcus pyogenes (yellow) is targeted to genomic DNA by asynthetic guide RNA (sgRNA) consisting of a 20-nt guide sequence (blue)and a scaffold (red). The guide sequence base-pairs with the DNA target(blue), directly upstream of a requisite 5′-NGG protospacer adjacentmotif (PAM; magenta), and Cas9 mediates a double-stranded break (DSB) ˜3bp upstream of the PAM (red triangle).

FIG. 2A-F shows an exemplary CRISPR system, a possible mechanism ofaction, an example adaptation for expression in eukaryotic cells, andresults of tests assessing nuclear localization and CRISPR activity.FIG. 2C discloses SEQ ID NOS 23-24, respectively, in order ofappearance. FIG. 2E discloses SEQ ID NOS 25-27, respectively, in orderof appearance. FIG. 2F discloses SEQ ID NOS 28-32, respectively, inorder of appearance.

FIG. 3A-D shows results of an evaluation of SpCas9 specificity for anexample target. FIG. 3A discloses SEQ ID NOS 33, 26 and 34-44,respectively, in order of appearance. FIG. 3C discloses SEQ ID NO: 33.

FIG. 4A-G shows an exemplary vector system and results for its use indirecting homologous recombination in eukaryotic cells. FIG. 4Ediscloses SEQ ID NO: 45. FIG. 4F discloses SEQ ID NOS 46-47,respectively, in order of appearance. FIG. 4G discloses SEQ ID NOS48-52, respectively, in order of appearance.

FIG. 5 provides a table of protospacer sequences (SEQ ID NOS 16, 15, 14,53-58, 18, 17 and 59-63, respectively, in order of appearance) andsummarizes modification efficiency results for protospacer targetsdesigned based on exemplary S. pyogenes and S. thermophilus CRISPRsystems with corresponding PAMs against loci in human and mouse genomes.Cells were transfected with Cas9 and either pre-crRNA/tracrRNA orchimeric RNA, and analyzed 72 hours after transfection. Percent indelsare calculated based on Surveyor assay results from indicated cell lines(N=3 for all protospacer targets, errors are S.E.M., N.D. indicates notdetectable using the Surveyor assay, and N.T. indicates not tested inthis study).

FIG. 6A-C shows a comparison of different tracrRNA transcripts forCas9-mediated gene targeting. FIG. 6A discloses SEQ ID NOS 64-65,respectively, in order of appearance.

FIG. 7 shows a schematic of a surveyor nuclease assay for detection ofdouble strand break-induced micro-insertions and -deletions.

FIG. 8A-B shows exemplary bicistronic expression vectors for expressionof CRISPR system elements in eukaryotic cells. FIG. 8A discloses SEQ IDNOS 66-68, respectively, in order of appearance. FIG. 8B discloses SEQID NOS 69-71, respectively, in order of appearance.

FIG. 9A-C shows histograms of distances between adjacent S. pyogenesSF370 locus 1 PAM (NGG) (FIG. 9A) and S. thermophilus LMD9 locus 2 PAM(NNAGAAW) (FIG. 9B) in the human genome; and distances for each PAM bychromosome (Chr) (FIG. 9C).

FIG. 10A-D shows an exemplary CRISPR system, an example adaptation forexpression in eukaryotic cells, and results of tests assessing CRISPRactivity. FIG. 10B discloses SEQ ID NOS 72-73, respectively, in order ofappearance. FIG. 10C discloses SEQ ID NO: 74.

FIG. 11A-C shows exemplary manipulations of a CRISPR system fortargeting of genomic loci in mammalian cells. FIG. 11A discloses SEQ IDNO: 75. FIG. 11B discloses SEQ ID NOS 76-78, respectively, in order ofappearance.

FIG. 12A-B shows the results of a Northern blot analysis of crRNAprocessing in mammalian cells. FIG. 12A discloses SEQ ID NO: 79.

FIG. 13A-B shows an exemplary selection of protospacers in the humanPVALB and mouse Th loci. FIG. 13A discloses SEQ ID NO: 80. FIG. 13Bdiscloses SEQ ID NO: 81.

FIG. 14 shows example protospacer and corresponding PAM sequence targetsof the S. thermophilus CRISPR system in the human EMX1 locus. FIG. 14discloses SEQ ID NO: 74.

FIG. 15 provides a table of sequences (SEQ ID NOS 82-93, respectively,in order of appearance) for primers and probes used for Surveyor, RFLP,genomic sequencing, and Northern blot assays.

FIG. 16A-C shows exemplary manipulation of a CRISPR system with chimericRNAs and results of SURVEYOR assays for system activity in eukaryoticcells. FIG. 16A discloses SEQ ID NO: 94.

FIG. 17A-B shows a graphical representation of the results of SURVEYORassays for CRISPR system activity in eukaryotic cells.

FIG. 18 shows an exemplary visualization of some S. pyogenes Cas9 targetsites in the human genome using the UCSC genome browser. FIG. 18discloses SEQ ID NOS 95-173, respectively, in order of appearance.

FIG. 19A-D shows a circular depiction of the phylogenetic analysisrevealing five families of Cas9s, including three groups of large Cas9s(˜1400 amino acids) and two of small Cas9s (˜1100 amino acids).

FIG. 20A-F shows the linear depiction of the phylogenetic analysisrevealing five families of Cas9s, including three groups of large Cas9s(˜1400 amino acids) and two of small Cas9s (˜1100 amino acids).

FIG. 21A-D shows genome editing via homologous recombination. (a)Schematic of SpCas9 nickase, with D10A mutation in the RuvC I catalyticdomain. (b) Schematic representing homologous recombination (HR) at thehuman EMX1 locus using either sense or antisense single strandedoligonucleotides as repair templates. Red arrow above indicates sgRNAcleavage site; PCR primers for genotyping (Tables J and K) are indicatedas arrows in right panel. FIG. 21C discloses SEQ ID NOS174-176, 174, 177and 176, respectively, in order of appearance. (c) Sequence of regionmodified by HR. d, SURVEYOR assay for wildtype (wt) and nickase (D10A)SpCas9-mediated indels at the EMX1 target 1 locus (n=3). Arrows indicatepositions of expected fragment sizes.

FIG. 22A-B shows single vector designs for SpCas9. FIG. 22A disclosesSEQ ID NOS 178-180, respectively, in order of appearance. FIG. 22Bdiscloses SEQ ID NO: 181.

The figures herein are for illustrative purposes only and are notnecessarily drawn to scale.

DETAILED DESCRIPTION OF THE INVENTION

The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”,“nucleic acid” and “oligonucleotide” are used interchangeably. Theyrefer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three dimensional structure, and mayperform any function, known or unknown. The following are non limitingexamples of polynucleotides: coding or non-coding regions of a gene orgene fragment, loci (locus) defined from linkage analysis, exons,introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, shortinterfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA),ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides,plasmids, vectors, isolated DNA of any sequence, isolated RNA of anysequence, nucleic acid probes, and primers. A polynucleotide maycomprise one or more modified nucleotides, such as methylatednucleotides and nucleotide analogs. If present, modifications to thenucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted by nonnucleotide components. A polynucleotide may be further modified afterpolymerization, such as by conjugation with a labeling component.

In aspects of the invention the terms “chimeric RNA”, “chimeric guideRNA”, “guide RNA”, “single guide RNA” and “synthetic guide RNA” are usedinterchangeably and refer to the polynucleotide sequence comprising theguide sequence, the tracr sequence and the tracr mate sequence. The term“guide sequence” refers to the about 20 bp sequence within the guide RNAthat specifies the target site and may be used interchangeably with theterms “guide” or “spacer”. The term “tracr mate sequence” may also beused interchangeably with the term “direct repeat(s)”. An exemplaryCRISPR-Cas system is illustrated in FIG. 1.

As used herein the term “wild type” is a term of the art understood byskilled persons and means the typical form of an organism, strain, geneor characteristic as it occurs in nature as distinguished from mutant orvariant forms.

As used herein the term “variant” should be taken to mean the exhibitionof qualities that have a pattern that deviates from what occurs innature.

The terms “non-naturally occurring” or “engineered” are usedinterchangeably and indicate the involvement of the hand of man. Theterms, when referring to nucleic acid molecules or polypeptides meanthat the nucleic acid molecule or the polypeptide is at leastsubstantially free from at least one other component with which they arenaturally associated in nature and as found in nature.

“Complementarity” refers to the ability of a nucleic acid to formhydrogen bond(s) with another nucleic acid sequence by eithertraditional Watson-Crick or other non-traditional types. A percentcomplementarity indicates the percentage of residues in a nucleic acidmolecule which can form hydrogen bonds (e.g., Watson-Crick base pairing)with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10being 50%, 60%, 70%, 80%, 90%, and 100% complementary). “Perfectlycomplementary” means that all the contiguous residues of a nucleic acidsequence will hydrogen bond with the same number of contiguous residuesin a second nucleic acid sequence. “Substantially complementary” as usedherein refers to a degree of complementarity that is at least 60%, 65%,70%, 75%, 80%, 85%, 90%. 95%, 97%, 98%, 99%, or 100% over a region of 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids thathybridize under stringent conditions.

As used herein, “stringent conditions” for hybridization refer toconditions under which a nucleic acid having complementarity to a targetsequence predominantly hybridizes with the target sequence, andsubstantially does not hybridize to non-target sequences. Stringentconditions are generally sequence-dependent, and vary depending on anumber of factors. In general, the longer the sequence, the higher thetemperature at which the sequence specifically hybridizes to its targetsequence. Non-limiting examples of stringent conditions are described indetail in Tijssen (1993), Laboratory Techniques In Biochemistry AndMolecular Biology-Hybridization With Nucleic Acid Probes Part I, SecondChapter “Overview of principles of hybridization and the strategy ofnucleic acid probe assay”, Elsevier, N.Y.

“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson Crick base pairing, Hoogstein binding, or inany other sequence specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming a multistranded complex, a single self hybridizing strand, or any combinationof these. A hybridization reaction may constitute a step in a moreextensive process, such as the initiation of PCR, or the cleavage of apolynucleotide by an enzyme. A sequence capable of hybridizing with agiven sequence is referred to as the “complement” of the given sequence.

As used herein, “expression” refers to the process by which apolynucleotide is transcribed from a DNA template (such as into and mRNAor other RNA transcript) and/or the process by which a transcribed mRNAis subsequently translated into peptides, polypeptides, or proteins.Transcripts and encoded polypeptides may be collectively referred to as“gene product.” If the polynucleotide is derived from genomic DNA,expression may include splicing of the mRNA in a eukaryotic cell.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non amino acids. The termsalso encompass an amino acid polymer that has been modified; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation, such asconjugation with a labeling component. As used herein the term “aminoacid” includes natural and/or unnatural or synthetic amino acids,including glycine and both the D or L optical isomers, and amino acidanalogs and peptidomimetics.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a vertebrate, preferably a mammal,more preferably a human. Mammals include, but are not limited to,murines, simians, humans, farm animals, sport animals, and pets.Tissues, cells and their progeny of a biological entity obtained in vivoor cultured in vitro are also encompassed.

The terms “therapeutic agent”, “therapeutic capable agent” or “treatmentagent” are used interchangeably and refer to a molecule or compound thatconfers some beneficial effect upon administration to a subject. Thebeneficial effect includes enablement of diagnostic determinations;amelioration of a disease, symptom, disorder, or pathological condition;reducing or preventing the onset of a disease, symptom, disorder orcondition; and generally counteracting a disease, symptom, disorder orpathological condition.

As used herein, “treatment” or “treating,” or “palliating” or“ameliorating” are used interchangeably. These terms refer to anapproach for obtaining beneficial or desired results including but notlimited to a therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant any therapeutically relevant improvement inor effect on one or more diseases, conditions, or symptoms undertreatment. For prophylactic benefit, the compositions may beadministered to a subject at risk of developing a particular disease,condition, or symptom, or to a subject reporting one or more of thephysiological symptoms of a disease, even though the disease, condition,or symptom may not have yet been manifested.

The term “effective amount” or “therapeutically effective amount” refersto the amount of an agent that is sufficient to effect beneficial ordesired results. The therapeutically effective amount may vary dependingupon one or more of: the subject and disease condition being treated,the weight and age of the subject, the severity of the diseasecondition, the manner of administration and the like, which can readilybe determined by one of ordinary skill in the art. The term also appliesto a dose that will provide an image for detection by any one of theimaging methods described herein. The specific dose may vary dependingon one or more of: the particular agent chosen, the dosing regimen to befollowed, whether it is administered in combination with othercompounds, timing of administration, the tissue to be imaged, and thephysical delivery system in which it is carried.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of immunology, biochemistry,chemistry, molecular biology, microbiology, cell biology, genomics andrecombinant DNA, which are within the skill of the art. See Sambrook,Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2ndedition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel,et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press,Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, ALABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).

Several aspects of the invention relate to vector systems comprising oneor more vectors, or vectors as such. Vectors can be designed forexpression of CRISPR transcripts (e.g. nucleic acid transcripts,proteins, or enzymes) in prokaryotic or eukaryotic cells. For example,CRISPR transcripts can be expressed in bacterial cells such asEscherichia coli, insect cells (using baculovirus expression vectors),yeast cells, or mammalian cells. Suitable host cells are discussedfurther in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY185, Academic Press. San Diego, Calif. (1990). Alternatively, therecombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Vectors may be introduced and propagated in a prokaryote. In someembodiments, a prokaryote is used to amplify copies of a vector to beintroduced into a eukaryotic cell or as an intermediate vector in theproduction of a vector to be introduced into a eukaryotic cell (e.g.amplifying a plasmid as part of a viral vector packaging system). Insome embodiments, a prokaryote is used to amplify copies of a vector andexpress one or more nucleic acids, such as to provide a source of one ormore proteins for delivery to a host cell or host organism. Expressionof proteins in prokaryotes is most often carried out in Escherichia coliwith vectors containing constitutive or inducible promoters directingthe expression of either fusion or non-fusion proteins. Fusion vectorsadd a number of amino acids to a protein encoded therein, such as to theamino terminus of the recombinant protein. Such fusion vectors may serveone or more purposes, such as: (i) to increase expression of recombinantprotein; (ii) to increase the solubility of the recombinant protein; and(iii) to aid in the purification of the recombinant protein by acting asa ligand in affinity purification. Often, in fusion expression vectors,a proteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase. Example fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,Academic Press, San Diego, Calif. (1990) 60-89).

In some embodiments, a vector is a yeast expression vector. Examples ofvectors for expression in yeast Saccharomyces cerivisae include pYepSec1(Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kuijan andHerskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), andpicZ (InVitrogen Corp, San Diego, Calif.).

In some embodiments, a vector drives protein expression in insect cellsusing baculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., SF9 cells)include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39).

In some embodiments, a vector is capable of driving expression of one ormore sequences in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, 1987.Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195).When used in mammalian cells, the expression vector's control functionsare typically provided by one or more regulatory elements. For example,commonly used promoters are derived from polyoma, adenovirus 2,cytomegalovirus, simian virus 40, and others disclosed herein and knownin the art. For other suitable expression systems for both prokaryoticand eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al.,MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989.

In some embodiments, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert, et al.,1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame andEaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) andimmunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen andBaltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci.USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985.Science 230: 912-916), and mammary gland-specific promoters (e.g., milkwhey promoter, U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990.Science 249: 374-379) and the α-fetoprotein promoter (Campes andTilghman, 1989. Genes Dev. 3: 537-546).

In some embodiments, a regulatory element is operably linked to one ormore elements of a CRISPR system so as to drive expression of the one ormore elements of the CRISPR system. In general, CRISPRs (ClusteredRegularly Interspaced Short Palindromic Repeats), also known as SPIDRs(SPacer Interspersed Direct Repeats), constitute a family of DNA locithat are usually specific to a particular bacterial species. The CRISPRlocus comprises a distinct class of interspersed short sequence repeats(SSRs) that were recognized in E. coli (Ishino et al., J. Bacteriol.,169:5429-5433 [1987]; and Nakata et al., J. Bacteriol., 171:3553-3556[1989]), and associated genes. Similar interspersed SSRs have beenidentified in Haloferax mediterranei, Streptococcus pyogenes, Anabaena,and Mycobacterium tuberculosis (See, Groenen et al., Mol. Microbiol.,10:1057-1065 [1993]; Hoe et al., Emerg. Infect. Dis., 5:254-263 [1999];Masepohl et al., Biochim. Biophys. Acta 1307:26-30 [1996]; and Mojica etal., Mol. Microbiol., 17:85-93 [1995]). The CRISPR loci typically differfrom other SSRs by the structure of the repeats, which have been termedshort regularly spaced repeats (SRSRs) (Janssen et al., OMICS J. Integ.Biol., 6:23-33 [2002]; and Mojica et al., Mol. Microbiol., 36:244-246[2000]). In general, the repeats are short elements that occur inclusters that are regularly spaced by unique intervening sequences witha substantially constant length (Mojica et al., [2000], supra). Althoughthe repeat sequences are highly conserved between strains, the number ofinterspersed repeats and the sequences of the spacer regions typicallydiffer from strain to strain (van Embden et al., J. Bacteriol.,182:2393-2401 [2000]). CRISPR loci have been identified in more than 40prokaryotes (See e.g., Jansen et al., Mol. Microbiol., 43:1565-1575[2002]; and Mojica et al., [2005]) including, but not limited toAeropyvrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula,Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus,Pyrococcus, Picrophilus, Thermoplasma, Corynebacteriunm, Mvcobacteriunm,Streptomyces, Aquifex, Porphvromonas, Chlorobium, Thermus, Bacillus,Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma,Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas,Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella,Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus,Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia,Treponema, and Thermotoga.

In general, “CRISPR system” refers collectively to transcripts and otherelements involved in the expression of or directing the activity ofCRISPR-associated (“Cas”) genes, including sequences encoding a Casgene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or anactive partial tracrRNA), a tracr-mate sequence (encompassing a “directrepeat” and a tracrRNA-processed partial direct repeat in the context ofan endogenous CRISPR system), a guide sequence (also referred to as a“spacer” in the context of an endogenous CRISPR system), or othersequences and transcripts from a CRISPR locus. In some embodiments, oneor more elements of a CRISPR system is derived from a type I, type II,or type III CRISPR system. In some embodiments, one or more elements ofa CRISPR system is derived from a particular organism comprising anendogenous CRISPR system, such as Streptococcus pyogenes. In general, aCRISPR system is characterized by elements that promote the formation ofa CRISPR complex at the site of a target sequence (also referred to as aprotospacer in the context of an endogenous CRISPR system). In thecontext of formation of a CRISPR complex, “target sequence” refers to asequence to which a guide sequence is designed to have complementarity,where hybridization between a target sequence and a guide sequencepromotes the formation of a CRISPR complex. Full complementarity is notnecessarily required, provided there is sufficient complementarity tocause hybridization and promote formation of a CRISPR complex. A targetsequence may comprise any polynucleotide, such as DNA or RNApolynucleotides. In some embodiments, a target sequence is located inthe nucleus or cytoplasm of a cell. In some embodiments, the targetsequence may be within an organelle of a eukaryotic cell, for example,mitochondrion or chloroplast. A sequence or template that may be usedfor recombination into the targeted locus comprising the targetsequences is referred to as an “editing template” or “editingpolynucleotide” or “editing sequence”. In aspects of the invention, anexogenous template polynucleotide may be referred to as an editingtemplate. In an aspect of the invention the recombination is homologousrecombination.

Typically, in the context of an endogenous CRISPR system, formation of aCRISPR complex (comprising a guide sequence hybridized to a targetsequence and complexed with one or more Cas proteins) results incleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence.Without wishing to be bound by theory, the tracr sequence, which maycomprise or consist of all or a portion of a wild-type tracr sequence(e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, ormore nucleotides of a wild-type tracr sequence), may also form part of aCRISPR complex, such as by hybridization along at least a portion of thetracr sequence to all or a portion of a tracr mate sequence that isoperably linked to the guide sequence. In some embodiments, the tracrsequence has sufficient complementarity to a tracr mate sequence tohybridize and participate in formation of a CRISPR complex. As with thetarget sequence, it is believed that complete complementarity is notneeded, provided there is sufficient to be functional. In someembodiments, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%,95% or 99% of sequence complementarity along the length of the tracrmate sequence when optimally aligned. In some embodiments, one or morevectors driving expression of one or more elements of a CRISPR systemare introduced into a host cell such that expression of the elements ofthe CRISPR system direct formation of a CRISPR complex at one or moretarget sites. For example, a Cas enzyme, a guide sequence linked to atracr-mate sequence, and a tracr sequence could each be operably linkedto separate regulatory elements on separate vectors. Alternatively, twoor more of the elements expressed from the same or different regulatoryelements, may be combined in a single vector, with one or moreadditional vectors providing any components of the CRISPR system notincluded in the first vector. CRISPR system elements that are combinedin a single vector may be arranged in any suitable orientation, such asone element located 5′ with respect to (“upstream” of) or 3′ withrespect to (“downstream” of) a second element. The coding sequence ofone element may be located on the same or opposite strand of the codingsequence of a second element, and oriented in the same or oppositedirection. In some embodiments, a single promoter drives expression of atranscript encoding a CRISPR enzyme and one or more of the guidesequence, tracr mate sequence (optionally operably linked to the guidesequence), and a tracr sequence embedded within one or more intronsequences (e.g. each in a different intron, two or more in at least oneintron, or all in a single intron). In some embodiments, the CRISPRenzyme, guide sequence, tracr mate sequence, and tracr sequence areoperably linked to and expressed from the same promoter. Single vectorconstructs for SpCas9 are illustrated in FIG. 22.

In some embodiments, a vector comprises one or more insertion sites,such as a restriction endonuclease recognition sequence (also referredto as a “cloning site”). In some embodiments, one or more insertionsites (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore insertion sites) are located upstream and/or downstream of one ormore sequence elements of one or more vectors. In some embodiments, avector comprises an insertion site upstream of a tracr mate sequence,and optionally downstream of a regulatory element operably linked to thetracr mate sequence, such that following insertion of a guide sequenceinto the insertion site and upon expression the guide sequence directssequence-specific binding of a CRISPR complex to a target sequence in aeukaryotic cell. In some embodiments, a vector comprises two or moreinsertion sites, each insertion site being located between two tracrmate sequences so as to allow insertion of a guide sequence at eachsite. In such an arrangement, the two or more guide sequences maycomprise two or more copies of a single guide sequence, two or moredifferent guide sequences, or combinations of these. When multipledifferent guide sequences are used, a single expression construct may beused to target CRISPR activity to multiple different, correspondingtarget sequences within a cell. For example, a single vector maycomprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,or more guide sequences. In some embodiments, about or more than about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such guide-sequence-containingvectors may be provided, and optionally delivered to a cell.

In some embodiments, a vector comprises a regulatory element operablylinked to an enzyme-coding sequence encoding a CRISPR enzyme, such as aCas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B,Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 andCsx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2.Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2,Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2,Csf3, Csf4, homologs thereof, or modified versions thereof. Theseenzymes are known; for example, the amino acid sequence of S. pyogenesCas9 protein may be found in the SwissProt database under accessionnumber Q99ZW2. In some embodiments, the unmodified CRISPR enzyme has DNAcleavage activity, such as Cas9. In some embodiments the CRISPR enzymeis Cas9, and may be Cas9 from S. pyogenes or S. pneumoniae. In someembodiments, the CRISPR enzyme directs cleavage of one or both strandsat the location of a target sequence, such as within the target sequenceand/or within the complement of the target sequence. In someembodiments, the CRISPR enzyme directs cleavage of one or both strandswithin about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200,500, or more base pairs from the first or last nucleotide of a targetsequence. In some embodiments, a vector encodes a CRISPR enzyme that ismutated to with respect to a corresponding wild-type enzyme such thatthe mutated CRISPR enzyme lacks the ability to cleave one or bothstrands of a target polynucleotide containing a target sequence. Forexample, an aspartate-to-alanine substitution (D10A) in the RuvC Icatalytic domain of Cas9 from S. pyogenes converts Cas9 from a nucleasethat cleaves both strands to a nickase (cleaves a single strand). Otherexamples of mutations that render Cas9 a nickase include, withoutlimitation, H840A, N854A, and N863A. In aspects of the invention,nickases may be used for genome editing via homologous recombination,For example, FIG. 21 shows genome editing via homologous recombination.FIG. 21 (a) shows the schematic of SpCas9 nickase, with D10A mutation inthe RuvC I catalytic domain. (b) Schematic representing homologousrecombination (HR) at the human EMX1 locus using either sense orantisense single stranded oligonucleotides as repair templates. (c)Sequence of region modified by HR. d, SURVEYOR assay for wildtype (wt)and nickase (D10A) SpCas9-mediated indels at the EMX1 target 1 locus(n=3). Arrows indicate positions of expected fragment sizes.

In some embodiments, a Cas9 nickase may be used in combination withguide sequence(s), e.g., two guide sequences, which target respectivelysense and antisense strands of the DNA target. This combination allowsboth strands to be nicked and used to induce NHEJ. Applicants havedemonstrated (data not shown) the efficacy of two nickase targets (i.e.,sgRNAs targeted at the same location but to different strands of DNA) ininducing mutagenic NHEJ. A single nickase (Cas9-D10A with a singlesgRNA) is unable to induce NHEJ and create indels but Applicants haveshown that double nickase (Cas9-D10A and two sgRNAs targeted todifferent strands at the same location) can do so in human embryonicstem cells (hESCs). The efficiency is about 50% of nuclease (i.e.,regular Cas9 without D10 mutation) in hESCs.

As a further example, two or more catalytic domains of Cas9 (RuvC I,RuvC II, and RuvC III) may be mutated to produce a mutated Cas9substantially lacking all DNA cleavage activity. In some embodiments, aD10A mutation is combined with one or more of H840A, N854A, or N863Amutations to produce a Cas9 enzyme substantially lacking all DNAcleavage activity. In some embodiments, a CRISPR enzyme is considered tosubstantially lack all DNA cleavage activity when the DNA cleavageactivity of the mutated enzyme is less than about 25%, 10%, 5%, 1%,0.1%, 0.01%, or lower with respect to its non-mutated form. Othermutations may be useful; where the Cas9 or other CRISPR enzyme is from aspecies other than S. pyogenes, mutations in corresponding amino acidsmay be made to achieve similar effects.

In some embodiments, an enzyme coding sequence encoding a CRISPR enzymeis codon optimized for expression in particular cells, such aseukaryotic cells. The eukaryotic cells may be those of or derived from aparticular organism, such as a mammal, including but not limited tohuman, mouse, rat, rabbit, dog, or non-human primate. In general, codonoptimization refers to a process of modifying a nucleic acid sequencefor enhanced expression in the host cells of interest by replacing atleast one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15,20, 25, 50, or more codons) of the native sequence with codons that aremore frequently or most frequently used in the genes of that host cellwhile maintaining the native amino acid sequence. Various speciesexhibit particular bias for certain codons of a particular amino acid.Codon bias (differences in codon usage between organisms) oftencorrelates with the efficiency of translation of messenger RNA (mRNA),which is in turn believed to be dependent on, among other things, theproperties of the codons being translated and the availability ofparticular transfer RNA (tRNA) molecules. The predominance of selectedtRNAs in a cell is generally a reflection of the codons used mostfrequently in peptide synthesis. Accordingly, genes can be tailored foroptimal gene expression in a given organism based on codon optimization.Codon usage tables are readily available, for example, at the “CodonUsage Database”, and these tables can be adapted in a number of ways.See Nakamura, Y., et al. “Codon usage tabulated from the internationalDNA sequence databases: status for the year 2000” Nucl. Acids Res.28:292 (2000). Computer algorithms for codon optimizing a particularsequence for expression in a particular host cell are also available,such as Gene Forge (Aptagen; Jacobus, Pa.), are also available. In someembodiments, one or more codons (e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50,or more, or all codons) in a sequence encoding a CRISPR enzymecorrespond to the most frequently used codon for a particular aminoacid.

In general, a guide sequence is any polynucleotide sequence havingsufficient complementarity with a target polynucleotide sequence tohybridize with the target sequence and direct sequence-specific bindingof a CRISPR complex to the target sequence. In some embodiments, thedegree of complementarity between a guide sequence and its correspondingtarget sequence, when optimally aligned using a suitable alignmentalgorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%,95%, 97.5%, 99%, or more. Optimal alignment may be determined with theuse of any suitable algorithm for aligning sequences, non-limitingexample of which include the Smith-Waterman algorithm, theNeedleman-Wunsch algorithm, algorithms based on the Burrows-WheelerTransform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT,Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.),SOAP (available at soap.genomics.org.cn), and Maq (available atmaq.sourceforge.net). In some embodiments, a guide sequence is about ormore than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotidesin length. In some embodiments, a guide sequence is less than about 75,50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. Theability of a guide sequence to direct sequence-specific binding of aCRISPR complex to a target sequence may be assessed by any suitableassay. For example, the components of a CRISPR system sufficient to forma CRISPR complex, including the guide sequence to be tested, may beprovided to a host cell having the corresponding target sequence, suchas by transfection with vectors encoding the components of the CRISPRsequence, followed by an assessment of preferential cleavage within thetarget sequence, such as by Surveyor assay as described herein.Similarly, cleavage of a target polynucleotide sequence may be evaluatedin a test tube by providing the target sequence, components of a CRISPRcomplex, including the guide sequence to be tested and a control guidesequence different from the test guide sequence, and comparing bindingor rate of cleavage at the target sequence between the test and controlguide sequence reactions. Other assays are possible, and will occur tothose skilled in the art.

A guide sequence may be selected to target any target sequence. In someembodiments, the target sequence is a sequence within a genome of acell. Exemplary target sequences include those that are unique in thetarget genome. For example, for the S. pyogenes Cas9, a unique targetsequence in a genome may include a Cas9 target site of the formMMMMMMMMNNNNNNNNNNNNXGG where NNNNNNNNNNNNXGG (N is A, G, T, or C; and Xcan be anything) has a single occurrence in the genome. A unique targetsequence in a genome may include an S. pyogenes Cas9 target site of theform MMMMMMMMMNNNNNNNNNNNXGG where NNNNNNNNNNNXGG (N is A, G, T, or C;and X can be anything) has a single occurrence in the genome. For the S.thermophilus CRISPR1Cas9, a unique target sequence in a genome mayinclude a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXXAGAAW (SEQID NO: 1) where NNNNNNNNNNNNXXAGAAW (SEQ ID NO: 2) (N is A, G, T, or C;X can be anything; and W is A or T) has a single occurrence in thegenome. A unique target sequence in a genome may include an S.thermophilus CRISPR 1 Cas9 target site of the formMMMMMMMMMNNNNNNNNNNNXXAGAAW (SEQ ID NO: 3) where NNNNNNNNNNNXXAGAAW (SEQID NO: 4) (N is A, G, T, or C; X can be anything; and W is A or T) has asingle occurrence in the genome. For the S. pyogenes Cas9, a uniquetarget sequence in a genome may include a Cas9 target site of the formMMMMMMMMNNNNNNNNNNNNXGGXG where NNNNNNNNNNNNXGGXG (N is A, G, T, or C;and X can be anything) has a single occurrence in the genome. A uniquetarget sequence in a genome may include an S. pyogenes Cas9 target siteof the form MMMMMMMMMNNNNNNNNNNNXGGXG where NNNNNNNNNNNXGGXG (N is A, G,T, or C; and X can be anything) has a single occurrence in the genome.In each of these sequences “M” may be A, G, T, or C, and need not beconsidered in identifying a sequence as unique.

In some embodiments, a guide sequence is selected to reduce the degreeof secondary structure within the guide sequence. Secondary structuremay be determined by any suitable polynucleotide folding algorithm. Someprograms are based on calculating the minimal Gibbs free energy. Anexample of one such algorithm is mFold, as described by Zuker andStiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example foldingalgorithm is the online webserver RNAfold, developed at Institute forTheoretical Chemistry at the University of Vienna, using the centroidstructure prediction algorithm (see e.g. A. R. Gruber et al., 2008, Cell106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology27(12): 1151-62). Further algorithms may be found in U.S. applicationSer. No. 61/836,080; Broad Reference BI-2013/004A); incorporated hereinby reference.

In general, a tracr mate sequence includes any sequence that hassufficient complementarity with a tracr sequence to promote one or moreof: (1) excision of a guide sequence flanked by tracr mate sequences ina cell containing the corresponding tracr sequence; and (2) formation ofa CRISPR complex at a target sequence, wherein the CRISPR complexcomprises the tracr mate sequence hybridized to the tracr sequence. Ingeneral, degree of complementarity is with reference to the optimalalignment of the tracr mate sequence and tracr sequence, along thelength of the shorter of the two sequences. Optimal alignment may bedetermined by any suitable alignment algorithm, and may further accountfor secondary structures, such as self-complementarity within either thetracr sequence or tracr mate sequence. In some embodiments, the degreeof complementarity between the tracr sequence and tracr mate sequencealong the length of the shorter of the two when optimally aligned isabout or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,97.5%, 99%, or higher. Example illustrations of optimal alignmentbetween a tracr sequence and a tracr mate sequence are provided in FIGS.10B and 11B. In some embodiments, the tracr sequence is about or morethan about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 40, 50, or more nucleotides in length. In some embodiments, thetracr sequence and tracr mate sequence are contained within a singletranscript, such that hybridization between the two produces atranscript having a secondary structure, such as a hairpin. Preferredloop forming sequences for use in hairpin structures are fournucleotides in length, and most preferably have the sequence GAAA.However, longer or shorter loop sequences may be used, as mayalternative sequences. The sequences preferably include a nucleotidetriplet (for example, AAA), and an additional nucleotide (for example Cor G). Examples of loop forming sequences include CAAA and AAAG. In anembodiment of the invention, the transcript or transcribedpolynucleotide sequence has at least two or more hairpins. In preferredembodiments, the transcript has two, three, four or five hairpins. In afurther embodiment of the invention, the transcript has at most fivehairpins. In some embodiments, the single transcript further includes atranscription termination sequence; preferably this is a polyT sequence,for example six T nucleotides. An example illustration of such a hairpinstructure is provided in the lower portion of FIG. 11B, where theportion of the sequence 5′ of the final “N” and upstream of the loopcorresponds to the tracr mate sequence, and the portion of the sequence3′ of the loop corresponds to the tracr sequence. Further non-limitingexamples of single polynucleotides comprising a guide sequence, a tracrmate sequence, and a tracr sequence are as follows (listed 5′ to 3′),where “N” represents a base of a guide sequence, the first block oflower case letters represent the tracr mate sequence, and the secondblock of lower case letters represent the tracr sequence, and the finalpoly-T sequence represents the transcription terminator: (1)NNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO:5); (2)NNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 6); (3)NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgtTTTTT (SEQ ID NO: 7); (4)NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTTT (SEQ ID NO: 8); (5)NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaacttgaaaaagtgTTTTTTT (SEQ ID NO: 9); and (6)NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTTTTTTTT (SEQ ID NO: 10). In some embodiments, sequences (1) to (3) are usedin combination with Cas9 from S. thermophilus CRISPR1. In someembodiments, sequences (4) to (6) are used in combination with Cas9 fromS. pyogenes. In some embodiments, the tracr sequence is a separatetranscript from a transcript comprising the tracr mate sequence (such asillustrated in the top portion of FIG. 11B).

In some embodiments, the CRISPR enzyme is part of a fusion proteincomprising one or more heterologous protein domains (e.g. about or morethan about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition tothe CRISPR enzyme). A CRISPR enzyme fusion protein may comprise anyadditional protein sequence, and optionally a linker sequence betweenany two domains. Examples of protein domains that may be fused to aCRISPR enzyme include, without limitation, epitope tags, reporter genesequences, and protein domains having one or more of the followingactivities: methylase activity, demethylase activity, transcriptionactivation activity, transcription repression activity, transcriptionrelease factor activity, histone modification activity, RNA cleavageactivity and nucleic acid binding activity. Non-limiting examples ofepitope tags include histidine (His) tags, V5 tags, FLAG tags, influenzahemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx)tags. Examples of reporter genes include, but are not limited to,glutathione-5-transferase (GST), horseradish peroxidase (HRP),chloramphenicol acetyltransferase (CAT) beta-galactosidase,beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed,DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP),and autofluorescent proteins including blue fluorescent protein (BFP). ACRISPR enzyme may be fused to a gene sequence encoding a protein or afragment of a protein that bind DNA molecules or bind other cellularmolecules, including but not limited to maltose binding protein (MBP),S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domainfusions, and herpes simplex virus (HSV) BP16 protein fusions. Additionaldomains that may form part of a fusion protein comprising a CRISPRenzyme are described in US20110059502, incorporated herein by reference.In some embodiments, a tagged CRISPR enzyme is used to identify thelocation of a target sequence.

In an aspect of the invention, a reporter gene which includes but is notlimited to glutathione-5-transferase (GST), horseradish peroxidase(HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase,beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed,DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP),and autofluorescent proteins including blue fluorescent protein (BFP),may be introduced into a cell to encode a gene product which serves as amarker by which to measure the alteration or modification of expressionof the gene product. In a further embodiment of the invention, the DNAmolecule encoding the gene product may be introduced into the cell via avector. In a preferred embodiment of the invention the gene product isluciferase. In a further embodiment of the invention the expression ofthe gene product is decreased.

In some aspects, the invention provides methods comprising deliveringone or more polynucleotides, such as or one or more vectors as describedherein, one or more transcripts thereof, and/or one or proteinstranscribed therefrom, to a host cell. In some aspects, the inventionfurther provides cells produced by such methods, and organisms (such asanimals, plants, or fungi) comprising or produced from such cells. Insome embodiments, a CRISPR enzyme in combination with (and optionallycomplexed with) a guide sequence is delivered to a cell. Conventionalviral and non-viral based gene transfer methods can be used to introducenucleic acids in mammalian cells or target tissues. Such methods can beused to administer nucleic acids encoding components of a CRISPR systemto cells in culture, or in a host organism. Non-viral vector deliverysystems include DNA plasmids, RNA (e.g. a transcript of a vectordescribed herein), naked nucleic acid, and nucleic acid complexed with adelivery vehicle, such as a liposome. Viral vector delivery systemsinclude DNA and RNA viruses, which have either episomal or integratedgenomes after delivery to the cell. For a review of gene therapyprocedures, see Anderson, Science 256:808-813 (1992); Nabel & Felgner,TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993);Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992);Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, RestorativeNeurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, BritishMedical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topicsin Microbiology and Immunology Doerfler and Bihm (eds) (1995); and Yu etal., Gene Therapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids include lipofection,nucleofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Lipofection isdescribed in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355)and lipofection reagents are sold commercially (e.g., Transfectam™ andLipofectin™). Cationic and neutral lipids that are suitable forefficient receptor-recognition lipofection of polynucleotides includethose of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells(e.g. in vitro or ex vivo administration) or target tissues (e.g. invivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleicacids take advantage of highly evolved processes for targeting a virusto specific cells in the body and trafficking the viral payload to thenucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro, and the modifiedcells may optionally be administered to patients (ex vivo). Conventionalviral based systems could include retroviral, lentivirus, adenoviral,adeno-associated and herpes simplex virus vectors for gene transfer.Integration in the host genome is possible with the retrovirus,lentivirus, and adeno-associated virus gene transfer methods, oftenresulting in long term expression of the inserted transgene.Additionally, high transduction efficiencies have been observed in manydifferent cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),Simian Immuno deficiency virus (SIV), human immuno deficiency virus(HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700). In applications where transient expression ispreferred, adenoviral based systems may be used. Adenoviral basedvectors are capable of very high transduction efficiency in many celltypes and do not require cell division. With such vectors, high titerand levels of expression have been obtained. This vector can be producedin large quantities in a relatively simple system. Adeno-associatedvirus (“AAV”) vectors may also be used to transduce cells with targetnucleic acids, e.g., in the in vitro production of nucleic acids andpeptides, and for in vivo and ex vivo gene therapy procedures (see,e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368;WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J.Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectorsare described in a number of publications, including U.S. Pat. No.5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985);Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat &Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol.63:03822-3828 (1989).

Packaging cells are typically used to form virus particles that arecapable of infecting a host cell. Such cells include 293 cells, whichpackage adenovirus, and ψ2 cells or PA317 cells, which packageretrovirus. Viral vectors used in gene therapy are usually generated byproducing a cell line that packages a nucleic acid vector into a viralparticle. The vectors typically contain the minimal viral sequencesrequired for packaging and subsequent integration into a host, otherviral sequences being replaced by an expression cassette for thepolynucleotide(s) to be expressed. The missing viral functions aretypically supplied in trans by the packaging cell line. For example, AAVvectors used in gene therapy typically only possess ITR sequences fromthe AAV genome which are required for packaging and integration into thehost genome. Viral DNA is packaged in a cell line, which contains ahelper plasmid encoding the other AAV genes, namely rep and cap, butlacking ITR sequences. The cell line may also be infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV. Additionalmethods for the delivery of nucleic acids to cells are known to thoseskilled in the art. See, for example, US20030087817, incorporated hereinby reference.

In some embodiments, a host cell is transiently or non-transientlytransfected with one or more vectors described herein. In someembodiments, a cell is transfected as it naturally occurs in a subject.In some embodiments, a cell that is transfected is taken from a subject.In some embodiments, the cell is derived from cells taken from asubject, such as a cell line. A wide variety of cell lines for tissueculture are known in the art. Examples of cell lines include, but arenot limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1,Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1,CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480,SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55,Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E,MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss,3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T,3T3, 721, 9L, A2780, A2780ADR, A2780cis, A 172, A20, A253, A431, A-549,ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293. BxPC3.C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T,CHO Dhfr −/−, COR-L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7,COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3,EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa,Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812,KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231,MDA-MB-468, MDA-MB-435, MDCK II, MDCK 11, MOR/0.2R, MONO-MAC 6, MTD-1A,MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3,NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F,RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line,U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, andtransgenic varieties thereof. Cell lines are available from a variety ofsources known to those with skill in the art (see, e.g., the AmericanType Culture Collection (ATCC) (Manassus, Va.)). In some embodiments, acell transfected with one or more vectors described herein is used toestablish a new cell line comprising one or more vector-derivedsequences. In some embodiments, a cell transiently transfected with thecomponents of a CRISPR system as described herein (such as by transienttransfection of one or more vectors, or transfection with RNA), andmodified through the activity of a CRISPR complex, is used to establisha new cell line comprising cells containing the modification but lackingany other exogenous sequence. In some embodiments, cells transiently ornon-transiently transfected with one or more vectors described herein,or cell lines derived from such cells are used in assessing one or moretest compounds.

In some embodiments, one or more vectors described herein are used toproduce a non-human transgenic animal or transgenic plant. In someembodiments, the transgenic animal is a mammal, such as a mouse, rat, orrabbit. In certain embodiments, the organism or subject is a plant. Incertain embodiments, the organism or subject or plant is algae. Methodsfor producing transgenic plants and animals are known in the art, andgenerally begin with a method of cell transfection, such as describedherein. Transgenic animals are also provided, as are transgenic plants,especially crops and algae. The transgenic animal or plant may be usefulin applications outside of providing a disease model. These may includefood or feed production through expression of, for instance, higherprotein, carbohydrate, nutrient or vitamins levels than would normallybe seen in the wildtype. In this regard, transgenic plants, especiallypulses and tubers, and animals, especially mammals such as livestock(cows, sheep, goats and pigs), but also poultry and edible insects, arepreferred.

Transgenic algae or other plants such as rape may be particularly usefulin the production of vegetable oils or biofuels such as alcohols(especially methanol and ethanol), for instance. These may be engineeredto express or overexpress high levels of oil or alcohols for use in theoil or biofuel industries.

In one aspect, the invention provides for methods of modifying a targetpolynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or invitro. In some embodiments, the method comprises sampling a cell orpopulation of cells from a human or non-human animal or plant (includingmicro-algae), and modifying the cell or cells. Culturing may occur atany stage ex vivo. The cell or cells may even be re-introduced into thenon-human animal or plant (including micro-algae).

In one aspect, the invention provides for methods of modifying a targetpolynucleotide in a eukaryotic cell. In some embodiments, the methodcomprises allowing a CRISPR complex to bind to the target polynucleotideto effect cleavage of said target polynucleotide thereby modifying thetarget polynucleotide, wherein the CRISPR complex comprises a CRISPRenzyme complexed with a guide sequence hybridized to a target sequencewithin said target polynucleotide, wherein said guide sequence is linkedto a tracr mate sequence which in turn hybridizes to a tracr sequence.

In one aspect, the invention provides a method of modifying expressionof a polynucleotide in a eukaryotic cell. In some embodiments, themethod comprises allowing a CRISPR complex to bind to the polynucleotidesuch that said binding results in increased or decreased expression ofsaid polynucleotide; wherein the CRISPR complex comprises a CRISPRenzyme complexed with a guide sequence hybridized to a target sequencewithin said polynucleotide, wherein said guide sequence is linked to atracr mate sequence which in turn hybridizes to a tracr sequence.

With recent advances in crop genomics, the ability to use CRISPR-Cassystems to perform efficient and cost effective gene editing andmanipulation will allow the rapid selection and comparison of single andmultiplexed genetic manipulations to transform such genomes for improvedproduction and enhanced traits. In this regard reference is made to USpatents and publications: U.S. Pat. No. 6,603,061—Agrobacterium-MediatedPlant Transformation Method; U.S. Pat. No. 7,868,149—Plant GenomeSequences and Uses Thereof and US 2009/0100536—Transgenic Plants withEnhanced Agronomic Traits, all the contents and disclosure of each ofwhich are herein incorporated by reference in their entirety. In thepractice of the invention, the contents and disclosure of Morrell et al“Crop genomics:advances and applications” Nat Rev Genet. 2011 Dec. 29;13(2):85-96 are also herein incorporated by reference in their entirety.

In plants, pathogens are often host-specific. For example, Fusariumoxysporum f. sp. lycopersici causes tomato wilt but attacks only tomato,and F. oxysporum f: dianthii Puccinia graminis f. sp. tritici attacksonly wheat. Plants have existing and induced defenses to resist mostpathogens. Mutations and recombination events across plant generationslead to genetic variability that gives rise to susceptibility,especially as pathogens reproduce with more frequency than plants. Inplants there can be non-host resistance, e.g., the host and pathogen areincompatible. There can also be Horizontal Resistance, e.g., partialresistance against all races of a pathogen, typically controlled by manygenes and Vertical Resistance, e.g., complete resistance to some racesof a pathogen but not to other races, typically controlled by a fewgenes. In a Gene-for-Gene level, plants and pathogens evolve together,and the genetic changes in one balance changes in other. Accordingly,using Natural Variability, breeders combine most useful genes for Yield,Quality, Uniformity, Hardiness, Resistance. The sources of resistancegenes include native or foreign Varieties, Heirloom Varieties, WildPlant Relatives, and Induced Mutations, e.g., treating plant materialwith mutagenic agents. Using the present invention, plant breeders areprovided with a new tool to induce mutations. Accordingly, one skilledin the art can analyze the genome of sources of resistance genes, and inVarieties having desired characteristics or traits employ the presentinvention to induce the rise of resistance genes, with more precisionthan previous mutagenic agents and hence accelerate and improve plantbreeding programs.

In one aspect, the invention provides kits containing any one or more ofthe elements disclosed in the above methods and compositions. In someembodiments, the kit comprises a vector system and instructions forusing the kit. In some embodiments, the vector system comprises (a) afirst regulatory element operably linked to a tracr mate sequence andone or more insertion sites for inserting a guide sequence upstream ofthe tracr mate sequence, wherein when expressed, the guide sequencedirects sequence-specific binding of a CRISPR complex to a targetsequence in a eukaryotic cell, wherein the CRISPR complex comprises aCRISPR enzyme complexed with (1) the guide sequence that is hybridizedto the target sequence, and (2) the tracr mate sequence that ishybridized to the tracr sequence; and/or (b) a second regulatory elementoperably linked to an enzyme-coding sequence encoding said CRISPR enzymecomprising a nuclear localization sequence. Elements may be providedindividually or in combinations, and may be provided in any suitablecontainer, such as a vial, a bottle, or a tube. In some embodiments, thekit includes instructions in one or more languages, for example in morethan one language.

In some embodiments, a kit comprises one or more reagents for use in aprocess utilizing one or more of the elements described herein. Reagentsmay be provided in any suitable container. For example, a kit mayprovide one or more reaction or storage buffers. Reagents may beprovided in a form that is usable in a particular assay, or in a formthat requires addition of one or more other components before use (e.g.in concentrate or lyophilized form). A buffer can be any buffer,including but not limited to a sodium carbonate buffer, a sodiumbicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, aHEPES buffer, and combinations thereof. In some embodiments, the bufferis alkaline. In some embodiments, the buffer has a pH from about 7 toabout 10. In some embodiments, the kit comprises one or moreoligonucleotides corresponding to a guide sequence for insertion into avector so as to operably link the guide sequence and a regulatoryelement. In some embodiments, the kit comprises a homologousrecombination template polynucleotide.

In one aspect, the invention provides methods for using one or moreelements of a CRISPR system. The CRISPR complex of the inventionprovides an effective means for modifying a target polynucleotide. TheCRISPR complex of the invention has a wide variety of utility includingmodifying (e.g., deleting, inserting, translocating, inactivating,activating) a target polynucleotide in a multiplicity of cell types. Assuch the CRISPR complex of the invention has a broad spectrum ofapplications in, e.g., gene therapy, drug screening, disease diagnosis,and prognosis. An exemplary CRISPR complex comprises a CRISPR enzymecomplexed with a guide sequence hybridized to a target sequence withinthe target polynucleotide. The guide sequence is linked to a tracr matesequence, which in turn hybridizes to a tracr sequence.

The target polynucleotide of a CRISPR complex can be any polynucleotideendogenous or exogenous to the eukaryotic cell. For example, the targetpolynucleotide can be a polynucleotide residing in the nucleus of theeukaryotic cell. The target polynucleotide can be a sequence coding agene product (e.g., a protein) or a non-coding sequence (e.g., aregulatory polynucleotide or a junk DNA). Without wishing to be bound bytheory, it is believed that the target sequence should be associatedwith a PAM (protospacer adjacent motif); that is, a short sequencerecognized by the CRISPR complex. The precise sequence and lengthrequirements for the PAM differ depending on the CRISPR enzyme used, butPAMs are typically 2-5 base pair sequences adjacent the protospacer(that is, the target sequence) Examples of PAM sequences are given inthe examples section below, and the skilled person will be able toidentify further PAM sequences for use with a given CRISPR enzyme.

The target polynucleotide of a CRISPR complex may include a number ofdisease-associated genes and polynucleotides as well as signalingbiochemical pathway-associated genes and polynucleotides as listed inU.S. provisional patent applications 61/736,527 and 61/748,427 havingBroad reference BI-2011/008/WSGR and BI-2011/008/WSGR respectively, bothentitled SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATIONfiled on Dec. 12, 2012 and Jan. 2, 2013, respectively, the contents ofall of which are herein incorporated by reference in their entirety.

Examples of target polynucleotides include a sequence associated with asignaling biochemical pathway, e.g., a signaling biochemicalpathway-associated gene or polynucleotide. Examples of targetpolynucleotides include a disease associated gene or polynucleotide. A“disease-associated” gene or polynucleotide refers to any gene orpolynucleotide which is yielding transcription or translation productsat an abnormal level or in an abnormal form in cells derived from adisease-affected tissues compared with tissues or cells of a non diseasecontrol. It may be a gene that becomes expressed at an abnormally highlevel; it may be a gene that becomes expressed at an abnormally lowlevel, where the altered expression correlates with the occurrenceand/or progression of the disease. A disease-associated gene also refersto a gene possessing mutation(s) or genetic variation that is directlyresponsible or is in linkage disequilibrium with a gene(s) that isresponsible for the etiology of a disease. The transcribed or translatedproducts may be known or unknown, and may be at a normal or abnormallevel.

Examples of disease-associated genes and polynucleotides are availablefrom McKusick-Nathans Institute of Genetic Medicine, Johns HopkinsUniversity (Baltimore, Md.) and National Center for BiotechnologyInformation, National Library of Medicine (Bethesda, Md.), available onthe World Wide Web.

Examples of disease-associated genes and polynucleotides are listed inTables A and B. Disease specific information is available fromMcKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University(Baltimore, Md.) and National Center for Biotechnology Information,National Library of Medicine (Bethesda, Md.), available on the WorldWide Web. Examples of signaling biochemical pathway-associated genes andpolynucleotides are listed in Table C.

Mutations in these genes and pathways can result in production ofimproper proteins or proteins in improper amounts which affect function.Further examples of genes, diseases and proteins are hereby incorporatedby reference from US Provisional application. Such genes, proteins andpathways may be the target polynucleotide of a CRISPR complex.

TABLE A DISEASE/DISORDERS GENE(S) Neoplasia PTEN; ATM; ATR; EGFR; ERBB2;ERBB3; ERBB4; Notch1; Notch2; Notch3; Notch4; AKT; AKT2; AKT3; HIF;HIF1a; HIF3a; Met; HRG; Bcl2; PPAR alpha; PPAR gamma; WT1 (Wilms Tumor);FGF Receptor Family members (5 members: 1, 2, 3, 4, 5); CDKN2a; APC; RB(retinoblastoma); MEN1; VHL; BRCA1; BRCA2; AR (Androgen Receptor);TSG101; IGF; IGF Receptor; Igfl (4 variants); Igf2 (3 variants); Igf 1Receptor; Igf 2 Receptor; Bax; Bcl2; caspases family (9 members: 1, 2,3, 4, 6, 7, 8, 9, 12); Kras; Apc Age-related Macular Aber; Ccl2; Cc2; cp(ceruloplasmin); Timp3; cathepsinD; Degeneration Vldlr; Ccr2Schizophrenia Neuregulin1 (Nrg1); Erb4 (receptor for Neuregulin);Complexin1 (Cplx1); Tph1 Tryptophan hydroxylase; Tph2 Tryptophanhydroxylase 2; Neurexin 1; GSK3; GSK3a; GSK3b Disorders 5-HTT (Slc6a4);COMT; DRD (Drd1a); SLC6A3; DAOA; DTNBP1; Dao (Dao1) Trinucleotide RepeatHTT (Huntington's Dx); SBMA/SMAX1/AR (Kennedy's Disorders Dx); FXN/X25(Friedrich's Ataxia); ATX3 (Machado- Joseph's Dx); ATXN1 and ATXN2(spinocerebellar ataxias); DMPK (myotonic dystrophy); Atrophin-1 andAtn1 (DRPLA Dx); CBP (Creb-BP-global instability); VLDLR (Alzheimer's);Atxn7; Atxn10 Fragile X Syndrome FMR2; FXR1; FXR2; mGLUR5 SecretaseRelated APH-1 (alpha and beta); Presenilin (Psen1); nicastrin Disorders(Ncstn); PEN-2 Others Nos1; Parp1; Nat1; Nat2 Prion-related disordersPrp ALS SOD1; ALS2; STEX; FUS; TARDBP; VEGF (VEGF-a; VEGF-b; VEGF-c)Drug addiction Prkce (alcohol); Drd2; Drd4; ABAT (alcohol); GRIA2; Grm5;Grin1; Htr1b; Grin2a; Drd3; Pdyn; Gria1 (alcohol) Autism Mecp2; BZRAP1;MDGA2; Sema5A; Neurexin 1; Fragile X (FMR2 (AFF2); FXR1; FXR2; Mglur5)Alzheimer's Disease E1; CHIP; UCH; UBB; Tau; LRP; PICALM; Clusterin;PS1; SORL1; CR1; Vldlr; Uba1; Uba3; CHIP28 (Aqp1, Aquaporin 1); Uchl1;Uchl3; APP Inflammation IL-10; IL-1 (IL-1a; IL-1b); IL-13; IL-17 (IL-17a(CTLA8); IL- 17b; IL-17c; IL-17d; IL-17f); II-23; Cx3cr1; ptpn22; TNFa;NOD2/CARD15 for IBD; IL-6; IL-12 (IL-12a; IL-12b); CTLA4; Cx3cl1Parkinson's Disease x-Synuclcin; DJ-1; LRRK2; Parkin; PINK1

TABLE B Blood and Anemia (CRAN1, CDA1, RPS19, DBA, PKLR, PK1, NT5C3,UMPH1, coagulation diseases PSN1, RHAG, RH50A, NRAMP2, SPTB, ALAS2,ANH1, ASB, and disorders ABCB7, ABC7, ASAT); Bare lymphocyte syndrome(TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11, MHC2TA, C2TA, RFX5, RFXAP,RFX5), Bleeding disorders (TBXA2R, P2RX1, P2X1); Factor H and factorH-like 1 (HF1, CFH, HUS); Factor V and factor VIII (MCFD2); Factor VIIdeficiency (F7); Factor X deficiency (F10); Factor XI deficiency (F11);Factor XII deficiency (F12, HAF); Factor XIIIA deficiency (F13A1, F13A);Factor XIIIB deficiency (F13B); Fanconi anemia (FANCA, FACA, FA1, FA,FAA, FAAP95, FAAP90, FLJ34064, FANCB, FANCC, FACC, BRCA2, FANCD1,FANCD2, FANCD, FACD, FAD, FANCE, FACE, FANCF, XRCC9, FANCG, BRIP1,BACH1, FANCJ, PHF9, FANCL, FANCM, KIAA1596); Hemophagocyticlymphohistiocytosis disorders (PRF1, HPLH2, UNC13D, MUNC13-4, HPLH3,HLH3, FHL3); Hemophilia A (F8, F8C, HEMA); Hemophilia B (F9, HEMB),Hemorrhagic disorders (PI, ATT, F5); Leukocyde deficiencies anddisorders (ITGB2, CD18, LCAMB, LAD, EIF2B1, EIF2BA, EIF2B2, EIF2B3,EIF2B5, LVWM, CACH, CLE, EIF2B4); Sickle cell anemia (HBB); Thalassemia(HBA2, HBB, HBD, LCRB, HBA1). Cell dysregulation B-cell non-Hodgkinlymphoma (BCL7A, BCL7); Leukemia (TAL1, and oncology TCL5, SCL, TAL2,FLT3, NBS1, NBS, ZNFN1A1, IK1, LYF1, diseases and disorders HOXD4,HOX4B, BCR, CML, PHL, ALL, ARNT, KRAS2, RASK2, GMPS, AF10, ARHGEF12,LARG, KIAA0382, CALM, CLTH, CEBPA, CEBP, CHIC2, BTL, FLT3, KIT, PBT,LPP, NPM1, NUP214, D9S46E, CAN, CAIN, RUNX1, CBFA2, AML1, WHSC1L1, NSD3,FLT3, AF1Q, NPM1, NUMA1, ZNF145, PLZF, PML, MYL, STAT5B, AF10, CALM,CLTH, ARL11, ARLTS1, P2RX7, P2X7, BCR, CML, PHL, ALL, GRAF, NF1, VRNF,WSS, NFNS, PTPN11, PTP2C, SHP2, NS1, BCL2, CCND1, PRAD1, BCL1, TCRA,GATA1, GF1, ERYF1, NFE1, ABL1, NQO1, DIA4, NMOR1, NUP214, D9S46E, CAN,CAIN). Inflammation and AIDS (KIR3DL1, NKAT3, NKB1, AMB11, K1R3DS1,IFNG, CXCL12, immune related SDF1); Autoimmune lymphoproliferativesyndrome (TNFRSF6, APT1, diseases and disorders FAS, CD95, ALPS1A);Combined immunodeficiency, (IL2RG, SCIDX1, SCIDX, IMD4); HIV-1 (CCL5,SCYA5, D17S136E, TCP228), HIV susceptibility or infection (IL10, CSIF,CMKBR2, CCR2, CMKBR5, CCCKR5 (CCR5)); Immunodeficiencies (CD3E, CD3G,AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSFS, CD40LG,HIGM1, IGM, FOXP3, IPEX, AIID, XPID, PIDX, TNFRSF14B, TACI; Inflammation(IL-10, IL-1 (IL-1a, IL-1b), IL-13, IL-17 (IL-17a (CTLA8), IL-17b,IL-17c, IL-17d, IL-17f), II-23, Cx3cr1, ptpn22, TNFa, NOD2/CARD15 forIBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cl1); Severe combinedimmunodeficiencies (SCIDs)(JAK3, JAKL, DCLRE1C, ARTEMIS, SCIDA, RAG1,RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG, SCIDX1, SCIDX,IMD4). Metabolic, liver, Amyloid neuropathy (TTR, PALB); Amyloidosis(APOA1, APP, AAA, kidney and protein CVAP, AD1, GSN, FGA, LYZ, TTR,PALB); Cirrhosis (KRT18, KRT8, diseases and disorders CIRH1A, NAIC,TEX292, KIAA1988); Cystic fibrosis (CFTR, ABCC7, CF, MRP7); Glycogenstorage diseases (SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, LAMP2, LAMPB,AGL, GDE, GBE1, GYS2, PYGL, PFKM); Hepatic adenoma, 142330 (TCF1, HNF1A,MODY3), Hepatic failure, early onset, and neurologic disorder (SCOD1,SCO1), Hepatic lipase deficiency (LIPC), Hepatoblastoma, cancer andcarcinomas (CTNNB1, PDGFRL, PDGRL, PRLTS, AXIN1, AXIN, CTNNB1, TP53,P53, LFS1, IGF2R, MPRI, MET, CASP8, MCH5; Medullary cystic kidneydisease (UMOD, HNFJ, FJHN, MCKD2, ADMCKD2); Phenylketonuria (PAH, PKU1,QDPR, DHPR, PTS); Polycystic kidney and hepatic disease (FCYT, PKHD1,ARPKD, PKD1, PKD2, PKD4, PKDTS, PRKCSH, G19P1, PCLD, SEC63).Muscular/Skeletal Becker muscular dystrophy (DMD, BMD, MYF6), DuchenneMuscular diseases and disorders Dystrophy (DMD, BMD); Emery-Dreifussmuscular dystrophy (LMNA, LMN1, EMD2, FPLD, CMD1A, HGPS, LGMD1B, LMNA,LMN1, EMD2, FPLD, CMD1A); Facioscapulohumeral muscular dystrophy(FSHMD1A, FSHD1A); Muscular dystrophy (FKRP, MDC1C, LGMD2I, LAMA2, LAMM,LARGE, KIAA0609, MDC1D, FCMD, TTID, MYOT, CAPN3, CANP3, DYSF, LGMD2B,SGCG, LGMD2C, DMDA1, SCG3, SGCA, ADL, DAG2, LGMD2D, DMDA2, SGCB, LGMD2E,SGCD, SGD, LGMD2F, CMD1L, TCAP, LGMD2G, CMD1N, TRIM32, HT2A, LGMD2H,FKRP, MDC1C, LCMD2I, TTN, CMD1G, TMD, LGMD2J, POMT1, CAV3, LGMD1C,SEPN1, SELN, RSMD1, PLEC1, PLTN, EBS1); Osteopetrosis (LRP5, BMND1,LRP7, LR3, OPPG, VBCH2, CLCN7, CLC7, OPTA2, OSTM1, GL, TCIRG1, TIRC7,OC116, OPTB1); Muscular atrophy (VAPB, VAPC, ALS8, SMN1, SMA1, SMA2,SMA3, SMA4, BSCL2, SPG17, GARS, SMAD1, CMT2D, HEXB, IGHMBP2, SMUBP2,CATF1, SMARD1). Neurological and ALS (SOD1, ALS2, STEX, FUS, TARDBP,VEGF (VEGF-a, VEGF-b, neuronal diseases and VEGF-c); Alzheimer disease(APP, AAA, CVAP, AD1, APOE, AD2, disorders PSEN2, AD4, STM2, APBB2,FE65L1, NOS3, PLAU, URK, ACE, DCP1, ACE1, MPO, PACIP1, PAXIP1L, PTIP,A2M, BLMH, BMH, PSEN1, AD3); Autism (Mecp2, BZRAP1, MDGA2, Sema5A,Neurexin 1, GLO1, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4,KIAA1260, AUTSX2); Fragile X Syndrome (FMR2, FXR1, FXR2, mGLUR5),Huntington's disease and disease like disorders (HD, IT15, PRNP, PRIP,JPH3, JP3, HDL2, TBP, SCA17); Parkinson disease (NR4A2, NURR1, NOT,TINUR, SNCAIP, TBP, SCA17, SNCA, NACP, PARK1, PARK4, DJ1, PARK7, LRRK2,PARK8, PINK1, PARK6, UCHL1, PARK5, SNCA, NACP, PARK1, PARK4, PRKN,PARK2, PDJ, DBH, NDUFV2); Rett syndrome (MECP2, RTT, PPMX, MRX16, MRX79,CDKL5, STK9, MECP2, RTT, PPMX, MRX16, MRX79, x-Synuclein, DJ-1);Schizophrenia (Neuregulin1 (Nrg1), Erb4 (receptor for Neuregulin),Complexin1 (Cplx1), Tph1 Tryptophan hydroxylase, Tph2, Tryptophanhydroxylase 2, Neurexin 1, GSK3, GSK3a, GSK3b, 5-HTT (Slc6a4), CONT, DRD(Drd1a), SLC6A3, DAOA, DTNBP1, Dao (Dao1)); Secretase Related Disorders(APH-1 (alpha and beta), Presenilin (Psen1), nicastrin, (Ncstn), PEN-2,Nos1, Parp1, Nat1, Nat2); Trinucleotide Repeat Disorders (HTT(Huntington's Dx), SBMA/SMAX1/AR (Kennedy's Dx), FXN/X25 (Friedrich'sAtaxia), ATX3 (Machado-Joseph's Dx), ATXN1 and ATXN2 (spinocerebellarataxias), DMPK (myotonic dystrophy), Atrophin-1 and Atn1 (DRPLA Dx), CBP(Creb-BP-global instability), VLDLR (Alzheimer's), Atxn7, Atxn10)Occular diseases and Age-related macular degeneration (Aber, Ccl2, Cc2,cp (ceruloplasmin), disorders Timp3, cathepsinD, Vldlr, Ccr2); Cataract(CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2, CP49, CP47, CRYAA, CRYA1,PAX6, AN2 MGDA, CRYBA1, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19, CRYGD,CRYG4,BFSP2, CP49, CP47, HSF4, CTM, HSF4, CTM, MIP, AQP0, CRYAB, CRYA2,CTPP2, CRYBB1, CRYGD, CRYG4, CRYBB2, CRYB2, CRYGC, CRYG3, CCL, CRYAA,CRYA1, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1);Corneal clouding and dystrophy (APOA1, TGFBI, CSD2, CDGG1, CSD, BIGH3,CDG2, TACSTD2, TROP2, M1S1, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD,PPCD2, PIP5K3, CFD); Cornea plana congenital (KERA, CNA2); Glaucoma(MYOC, TIGR, GLC1A, JOAG, GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1B1,GLC3A, OPA1, NTG, NPG, CYP1B1, GLC3A); Leber congenital amaurosis (CRB1,RP12, CRX, CORD2, CRD, RPGRIP1, LCA6, CORD9, RPE65, RP20, AIPL1, LCA4,GUCY2D, GUC2D, LCA1, CORD6, RDH12, LCA3); Macular dystrophy (ELOVL4,ADMD, STGD2, STGD3, RDS, RP7, PRPH2, PRPH, AVMD, AOFMD, VMD2). Epilepsy,myoclonic, EPM2A, MELF, EPM2 Lafora type, 254780 Epilepsy, myoclonic,NHLRC1, EPM2A, EPM2B Lafora type, 254780 Duchenne muscular DMD, BMDdystrophy, 310200 (3) AIDS, delayed/rapid KIR3DL1, NKAT3, NKB1, AMB11,progression to (3) KIR3DS1 AIDS, rapid IFNG progression to, 609423 (3)AIDS, resistance to CXXCL12, SDF1 (3) Alpha 1-Antitrypsin SERPINA1[serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, Deficiencyantitrypsin), member 1]; SERPINA2 [serpin peptidase inhibitor, clade A(alpha-1 antiproteinase, antitrypsin), member 2]; SERPINA3 [serpinpeptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin),member 3]; SERPINA5 [serpin peptidase inhibitor, clade A (alpha-1antiproteinase, antitrypsin), member 5]; SERPINA6 [serpin peptidaseinhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 6];SERPINA7 [serpin peptidase inhibitor, Glade A (alpha-1 antiproteinase,antitrypsin), member 7];″ AND ″SERPINA6 (serpin peptidase inhibitor,clade A (alpha-1 antiproteinase, antitrypsin), member 6)

TABLE C CELLULAR FUNCTION GENES PI3K/AKT Signaling PRKCE; ITGAM; ITGA5;IRAK1; PRKAA2; EIF2AK2; PTEN; EIF4E: PRKCZ; GRK6: MAPK1; TSC1; PLK1;AKT2; IKBKB; PIK3CA; CDK8; CDKN1B; NFKB2; BCL2; PIK3CB; PPP2R1A; MAPK8;BCL2L1; MAPK3; TSC2; ITGA1; KRAS; EIF4EBP1; RELA; PRKCD; NOS3; PRKAA1;MAPK9; CDK2; PPP2CA; PIM1; ITGB7; YWHAZ; ILK; TP53; RAF1; IKBKG; RELB;DYRK1A; CDKNIA; ITGB1; MAP2K2; JAK1; AKT1; JAK2; PIK3R1; CHUK; PDPK1;PPP2R5C; CTNNB1; MAP2K1; NFKB1; PAK3; ITGB3; CCND1; GSK3A; FRAP1; SFN;ITGA2; TTK; CSNK1A1; BRAF; GSK3B; AKT3; FOXO1; SGK; HSP90AA1; RPS6KB1ERK/MAPK Signaling PRKCE; ITGAM; ITGA5; HSPB1; IRAK1; PRKAA2; EIF2AK2;RAC1; RAP1A; TLN1; EIF4E; ELK1; GRK6; MAPK1; RAC2; PLK1; AKT2; PIK3CA;CDK8; CREB1; PRKC1; PTK2; FOS; RPS6KA4; PIK3CB; PPP2R1A; PIK3C3; MAPK8;MAPK3; ITGA1; ETS1; KRAS; MYCN; EIF4EBP1; PPARG; PRKCD; PRKAA1; MAPK9;SRC; CDK2; PPP2CA; PIM1; PIK3C2A; ITGB7; YWHAZ; PPP1CC; KSR1; PXN; RAF1;FYN; DYRK1A; ITGB1; MAP2K2; PAK4; PIK3R1; STAT3; PPP2R5C; MAP2K1; PAK3;ITGB3; ESR1; ITGA2; MYC; TTK; CSNK1A1; CRKL; BRAF; ATF4; PRKCA; SRF;STAT1; SGK Glucocorticoid Receptor RAC1; TAF4B; EP300; SMAD2; TRAF6;PCAF; ELK1; Signaling MAPKI; SMAD3; AKT2; 1KBKB; NCOR2; UBE2I; PIK3CA;CREB1; FOS; HSPA5; NFKB2; BCL2; MAP3K14; STAT5B; PIK3CB; PIK3C3; MAPK8;BCL2L1; MAPK3; TSC22D3; MAPK10; NRIP1; KRAS; MAPK13; RELA; STAT5A;MAPK9; NOS2A; PBX1; NR3C1; PIK3C2A; CDKN1C; TRAF2; SERPINE1; NCOA3;MAPK14; TNF; RAF1; IKBKG; MAP3K7; CREBBP; CDKN1A; MAP2K2; JAK1; IL8;NCOA2; AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1; NFKB1; TGFBR1; ESR1;SMAD4; CEBPB; JUN; AR; AKT3; CCL2; MMP1; STAT1; IL6; HSP90AA1 AxonalGuidance PRKCE; ITGAM; ROCK1; ITGA5; CXCR4; ADAM12; Signaling IGF1;RAC1; RAP1A; EIF4E; PRKCZ; NRP1; NTRK2; ARHGEF7; SMO; ROCK2; MAPK1; PGF;RAC2; PTPN11; GNAS; AKT2; PIK3CA; ERBB2; PRKCI; PTK2; CFL1; GNAQ;PIK3CB; CXCL12; PIK3C3; WNT11; PRKD1; GNB2L1; ABL1; MAPK3; ITGA1; KRAS;RHOA; PRKCD; PIK3C2A; ITGB7; GLI2; PXN; VASP; RAF1; FYN; ITGB1; MAP2K2;PAK4; ADAM17; AKT1; PIK3R1; GLI1; WNT5A; ADAM10; MAP2K1; PAK3; ITGB3;CDC42; VEGFA; ITGA2; EPHA8; CRKL; RND1; GSK3B; AKT3; PRKCA EphrinReceptor PRKCE; ITGAM; ROCK1; ITGA5; CXCR4; IRAK1; Signaling PRKAA2;EIF2AK2; RAC1; RAP1A; GRK6; ROCK2; MAPK1; PGF; RAC2; PTPN11; GNAS; PLK1;AKT2; DOK1; CDK8; CREB1; PTK2; CFL1; GNAQ; MAP3K14; CXCL12; MAPK8;GNB2L1; ABL1; MAPK3; ITGA1; KRAS; RHOA; PRKCD; PRKAA1; MAPK9; SRC; CDK2;PIM1; ITGB7; PXN; RAF1; FYN; DYRK1A; ITGB1; MAP2K2; PAK4, AKT1; JAK2;STAT3; ADAM10; MAP2K1; PAK3; ITGB3; CDC42; VEGFA; ITGA2; EPHA8; TTK;CSNK1A1; CRKL; BRAF; PTPN13; ATF4; AKT3; SGK Actin Cytoskeleton ACTN4;PRKCE; ITGAM; ROCK1; ITGA5; IRAK1; Signaling PRKAA2; EIF2AK2; RAC1; INS;ARHGEF7; GRK6; ROCK2; MAPK1; RAC2; PLK1; AKT2; PIK3CA; CDK8; PTK2; CPL1;PIK3CB; MYH9; DIAPH1; PIK3C3; MAPK8; F2R; MAPK3; SLC9A1; ITGA1; KRAS;RHOA; PRKCD; PRKAA1; MAPK9; CDK2; PIM1, PIK3C2A; ITGB7; PPP1CC; PXN;VIL2; RAF1; GSN; DYRK1A; ITGB1; MAP2K2; PAK4; PIP5K1A; PIK3R1; MAP2K1;PAK3; ITGB3; CDC42; APC; ITGA2; TTK; CSNK1A1; CRKL; BRAF; VAV3; SGKHuntington's Disease PRKCE; IGF1; EP300; RCOR1; PRKCZ; HDAC4; TGM2;Signaling MAPK1; CAPNS1; AKT2; EGFR; NCOR2; SP1; CAPN2; PIK3CA; HDAC5;CREB1; PRKCI; HSPA5; REST; GNAQ; PIK3CB; PIK3C3; MAPK8; IGF1R; PRKD1;GNB2L1; BCL2L1; CAPN1; MAPK3; CASP8; HDAC2; HDAC7A; PRKCD; HDAC11;MAPK9; HDAC9; PIK3C2A; HDAC3; TP53; CASP9; CREBBP; AKT1; PIK3R1; PDPK1;CASP1; APAF1; FRAP1; CASP2; JUN; BAX; ATF4; AKT3; PRKCA; CLTC; SGK;HDAC6; CASP3 Apoptosis Signaling PRKCE; ROCK1; BID; IRAK1; PRKAA2;EIF2AK2; BAK1; BIRC4; GRK6; MAPK1; CAPNS1; PLK1; AKT2; IKBKB; CAPN2;CDK8; FAS; NFKB2; BCL2; MAP3K14; MAPK8; BCL2L1; CAPN1; MAPK3; CASP8;KRAS; RELA; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; TP53; TNF; RAF1; IKBKG;RELB; CASP9; DYRK1A; MAP2K2; CHUK; APAF1; MAP2K1; NFKB1; PAK3; LMNA;CASP2; BIRC2; TTK; CSNKIA1; BRAF; BAX; PRKCA; SGK; CASP3; BIRC3; PARP1 BCell Receptor RAC1; PTEN; LYN; ELK1; MAPK1; RAC2; PTPN11; SignalingAKT2; IKBKB; PIK3CA; CREB1; SYK; NFKB2; CAMK2A; MAP3K14; PIK3CB; PIK3C3;MAPK8; BCL2L1; ABL1; MAPK3; ETS1; KRAS; MAPK13; RELA; PTPN6; MAPK9;EGR1; PIK3C2A; BTK; MAPK14; RAF1; IKBKG; RELB; MAP3K7; MAP2K2; AKT1;PIK3R1; CHUK; MAP2K1; NFKB1; CDC42; GSK3A; FRAP1; BCL6; BCL10; JUN;GSK3B; ATF4; AKT3; VAV3; RPS6KB1 Leukocyte Extravasation ACTN4; CD44;PRKCE; ITGAM; ROCK1; CXCR4; CYBA; Signaling RAC1; RAP1A; PRKCZ; ROCK2;RAC2; PTPN11; MMP14; PIK3CA; PRKCI; PTK2; PIK3CB; CXCL12; PIK3C3; MAPK8;PRKD1; ABL1; MAPK10; CYBB; MAPK13; RHOA; PRKCD; MAPK9; SRC; PIK3C2A;BTK; MAPK14; NOX1; PXN; VIL2; VASP; ITGB1; MAP2K2; CTNND1; PIK3R1;CTNNB1; CLDN1; CDC42; F11R; ITK; CRKL; VAV3; CTTN; PRKCA; MMP1; MMP9Integrin Signaling ACTN4; ITGAM; ROCK1; ITGA5; RAC1; PTEN; RAP1A; TLN1;ARHGEF7; MAPK1; RAC2; CAPNS1; AKT2; CAPN2; PIK3CA; PTK2; PIK3CB; PIK3C3;MAPK8; CAV1; CAPN1; ABL1; MAPK3; ITGA1; KRAS; RHOA; SRC; PIK3C2A; ITGB7;PPP1CC; ILK; PXN; VASP; RAF1; FYN; ITGB1; MAP2K2; PAK4; AKT1; PIK3R1;TNK2; MAP2K1; PAK3; ITGB3; CDC42; RND3; ITGA2; CRKL; BRAF; GSK3B; AKT3Acute Phase Response IRAK1; SOD2; MYD88; TRAF6; ELK1; MAPK1; PTPN11;Signaling AKT2; IKBKB; PIK3CA; FOS; NFKB2; MAP3K14; PIK3CB; MAPK8;RIPK1; MAPK3; IL6ST; KRAS; MAPK13; IL6R; RELA; SOCS1; MAPK9; FTL; NR3C1;TRAF2; SERPINE1; MAPK14; TNF; RAF1; PDK1; IKBKG; RELB; MAP3K7; MAP2K2;AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1; NFKB1; FRAP1; CEBPB; JUN; AKT3;IL1R1; IL6 PTEN Signaling ITGAM; ITGA5; RAC1; PTEN; PRKCZ; BCL2L11;MAPK1; RAC2; AKT2; EGFR; IKBKB; CBL; PIK3CA; CDKN1B; PTK2; NFKB2; BCL2;PIK3CB; BCL2L1; MAPK3; ITGA1; KRAS; ITGB7; ILK; PDGFRB; INSR; RAF1;IKBKG; CASP9; CDKN1A; ITGB1; MAP2K2; AKT1; PIK3R1; CHUK; PDGFRA; PDPK1;MAP2K1; NFKB1; ITGB3; CDC42; CCND1; GSK3A; ITGA2; GSK3B; AKT3; FOXO1;CASP3; RPS6KB1 p53 Signaling PTEN; EP300; BBC3; PCAF; FASN; BRCA1;GADD45A; BIRC5; AKT2; PIK3CA; CHEK1; TP53INP1; BCL2; PIK3CB; PIK3C3;MAPK8; THBS1; ATR; BCL2L1; E2F1; PMAIP1; CHEK2; TNFRSF10B; TP73; RB1;HDAC9; CDK2; PIK3C2A; MAPK14; TP53; LRDD; CDKN1A; HIPK2; AKT1; PIK3R1;RRM2B; APAF1; CTNNB1; SIRT1; CCND1; PRKDC; ATM; SFN; CDKN2A; JUN; SNAI2;GSK3B; BAX; AKT3 Aryl Hydrocarbon HSPR1; EP300; FASN; TGM2; RXRA; MAPK1;NQO1; Receptor Signaling NCOR2; SP1; ARNT; CDKN1B; FOS; CHEK1; SMARCA4;NEKB2; MAPK8; ALDH1A1; ATR; E2F1; MAPK3; NRIP1; CHEK2; RELA; TP73;GSTP1; RB1; SRC; CDK2; AHR; NFE2L2; NCOA3; TP53; TNF; CDKN1A; NCOA2;APAF1; NFKB1; CCND1; ATM; ESR1; CDKN2A; MYC; JUN; ESR2; BAX; IL6;CYP1B1; HSP90AA1 Xenobiotic Metabolism PRKCE; EP300; PRKCZ; RXRA; MAPK1;NQO1; Signaling NCOR2; PIK3CA; ARNT; PRKCI; NFKB2; CAMK2A; PIK3CB;PPP2R1A; PIK3C3; MAPK8; PRKD1; ALDH1A1; MAPK3; NRIP1; KRAS; MAPK13;PRKCD; GSTP1; MAPK9; NOS2A; ABCB1; AHR; PPP2CA; FTL; NFE2L2; PIK3C2A;PPARGC1A; MAPK14; TNF; RAF1; CREBBP; MAP2K2; PIK3R1; PPP2R5C; MAP2K1;NFKB1; KEAP1; PRKCA; EIF2AK3; IL6; CYP1B1; HSP90AA1 SAPK/JNK SignalingPRKCE; IRAK1; PRKAA2; EIF2AK2; RAC1; ELK1; GRK6; MAPK1; GADD45A; RAC2;PLK1; AKT2; PIK3CA; FADD; CDK8; PIK3CB; PIK3C3; MAPK8; RIPK1; GNB2L1;IRS1; MAPK3; MAPK10; DAXX; KRAS; PRKCD; PRKAA1; MAPK9; CDK2; PIM1;PIK3C2A; TRAF2; TP53; LCK; MAP3K7; DYRK1A; MAP2K2; PIK3R1; MAP2K1; PAK3;CDC42; JUN; TTK; CSNK1A1; CRKL; BRAF; SGK PPAr/RXR Signaling PRKAA2;EP300; INS; SMAD2; TRAF6; PPARA; FASN; RXRA; MAPK1; SMAD3; GNAS; IKBKB;NCOR2; ABCA1; GNAQ; NFKB2; MAP3K14; STAT5B; MAPK8; IRS1; MAPK3; KRAS;RELA; PRKAA1; PPARGC1A; NCOA3; MAPK14; INSR; RAF1; IKBKG; RELB; MAP3K7;CREBBP; MAP2K2; JAK2; CHUK; MAP2K1; NFKB1; TGFBR1; SMAD4; JUN; IL1R1;PRKCA; IL6; HSP90AA1; ADIPOQ NF-KB Signaling IRAK1; EIF2AK2; EP300; INS;MYD88; PRKCZ; TRAF6; TBK1; AKT2; EGFR; IKBKB; PIK3CA; BTRC; NFKB2;MAP3K14; PIK3CB; PIK3C3; MAPK8; RIPK1; HDAC2; KRAS; RELA; PIK3C2A;TRAF2; TLR4; PDGFRB; TNF; INSR; LCK; IKBKG; RELB; MAP3K7; CREBBP; AKT1;PIK3R1; CHUK; PDGFRA; NFKB1; TLR2; BCL10; GSK3B; AKT3; TNFAIP3; IL1R1Neuregulin Signaling ERBB4; PRKCE; ITGAM; ITGA5; PTEN; PRKCZ; ELK1;MAPK1; PTPN11; AKT2; EGFR; ERBB2; PRKCI; CDKN1B; STAT5B; PRKD1; MAPK3;ITGA1; KRAS; PRKCD; STAT5A; SRC; ITGB7; RAF1; ITGB1; MAP2K2; ADAM17;AKT1; PIK3R1; PDPK1; MAP2K1; ITGB3; EREG; FRAP1; PSEN1; ITGA2; MYC;NRG1; CRKL; AKT3; PRKCA; HSP90AA1; RPS6KB1 Wnt & Beta catenin CD44;EP300; LRP6; DVL3; CSNK1E; GJA1; SMO; Signaling AKT2; PIN1; CDH1; BTRC;GNAQ; MARK2; PPP2R1A; WNT11; SRC; DKK1; PPP2CA; SOX6; SFRP2; ILK; LEF1;SOX9; TP53; MAP3K7; CREBBP; TCF7L2; AKT1; PPP2R5C; WNT5A; LRP5; CTNNB1;TGFBR1; CCND1; GSK3A; DVL1; APC; CDKN2A; MYC; CSNK1A1; GSK3B; AKT3; SOX2Insulin Receptor PTEN; INS; EIF4E; PTPN1; PRKCZ; MAPK1; TSC1; SignalingPTPN11; AKT2; CBL; PIK3CA; PRKCI; PIK3CB; PIK3C3; MAPK8; IRS1; MAPK3;TSC2; KRAS; EIF4EBP1; SLC2A4; PIK3C2A; PPP1CC; INSR; RAF1; FYN; MAP2K2;JAK1; AKT1; JAK2; PIK3R1; PDPK1; MAP2K1; GSK3A; FRAP1; CRKL; GSK3B;AKT3; FOXO1; SGK; RPS6KB1 IL-6 Signaling HSPB1; TRAF6; MAPKAPK2; :ELK1;MAPK1; PTPN11; IKBKB; FOS; NFKB2; MAP3K14; MAPK8; MAPK3; MAPK10; IL6ST;KRAS; MAPK13; IL6R; RELA; SOCS1; MAPK9; ABCB1; TRAF2; MAPK14; TNF; RAF1;IKBKG; RELB; MAP3K7; MAP2K2, IL8; JAK2; CHUK; STAT3; MAP2K1; NFKB1;CEBPB; JUN; IL1R1; SRF; IL6 Hepatic Cholestasis PRKCE; IRAK1; INS;MYD88; PRKCZ; TRAF6; PPARA; RXRA; IKBKB; PRKCI; NFKB2; MAP3K14; MAPK8;PRKD1; MAPK10; RELA; PRKCD; MAPK9; ABCB1; TRAF2; TLR4; TNF; INSR; IKBKG,RELB; MAP3K7; IL8; CHUK; NR1H2; TJP2; NFKB1; ESR1; SREBF1; FGFR4; JUN;IL1R1; PRKCA; IL6 IGF-1 Signaling IGF1; PRKCZ; ELK1; MAPK1; PTPN11;NEDD4; AKT2; PIK3CA; PRKCI; PTK2; FOS; PIK3CB; PIK3C3; MAPK8; IGF1R;IRS1; MAPK3; IGFBP7; KRAS; PIK3C2A; YWHAZ; PXN; RAF1; CASP9; MAP2K2;AKT1; PIK3R1; PDPK1; MAP2K1; IGFBP2; SFN; JUN; CYR61; AKT3; FOXO1; SRF;CTGF; RPS6KB1 NRF2-mediated PRKCE; EP300; SOD2; PRKCZ; MAPK1; SQSTM1;Oxidative Stress Response NQO1; PIK3CA; PRKCI; FOS; PIK3CB; PIK3C3;MAPK8; PRKD1; MAPK3; KRAS; PRKCD; GSTP1; MAPK9; FTL; NFE2L2; PIK3C2A;MAPK14; RAF1; MAP3K7; CREBBP; MAP2K2; AKT1; PIK3R1; MAP2K1; PPIB; JUN;KEAP1; GSK3B; ATF4; PRKCA; EIF2AK3; HSP90AA1 Hepatic Fibrosis/HepaticEDN1; IGF1; KDR; FLT1; SMAD2; FGFR1; MET; PGF; Stellate Cell ActivationSMAD3; EGFR; FAS; CSF1; NFKB2; BCL2; MYH9; IGF1R; IL6R; RELA; TLR4;PDGFRB; TNF; RELB; IL8; PDGFRA; NFKB1; TGFBR1; SMAD4; VEGFA; BAX; IL1R1;CCL2; HGF; MMP1; STAT1; IL6; CTGF; MMP9 PPAR Signaling EP300; INS;TRAF6; PPARA; RXRA; MAPK1; IKBKB; NCOR2; FOS; NFKB2; MAP3K14; STAT5B;MAPK3; NRIP1; KRAS; PPARG; RELA; STAT5A; TRAF2; PPARGC1A; PDGFRB; TNF;INSR; RAF1; IKBKG RELB; MAP3K7; CREBBP; MAP2K2; CHUK; PDGFRA; MAP2K1;NFKB1; JUN; IL1R1; HSP90AA1 Fc Epsilon R1 Signaling PRKCE; RAC1; PRKCZ;LYN; MAPK1; RAC2; PTPN11; AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3;MAPK8; PRKD1; MAPK3; MAPK10; KRAS; MAPK13; PRKCD; MAPK9; PIK3C2A; BTK;MAPK14; TNF; RAF1; FYN; MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; AKT3; VAV3;PRKCA G-Protein Coupled PRKCE; RAP1A; RGS16; MAPK1; GNAS; AKT2; IKBKB;Receptor Signaling PIK3CA; CREB1; GNAQ; NFKB2; CAMK2A; PIK3CB; PIK3C3;MAPK3; KRAS; RELA; SRC; PIK3C2A; RAF1; IKBKG; RELB; FYN; MAP2K2; AKT1;PIK3R1; CHUK; PDPK1; STAT3; MAP2K1; NFKB1; BRAF; ATF4; AKT3; PRKCAInositol Phosphate PRKCE; IRAK1; PRKAA2; EIF2AK2; PTEN; GRK6; MetabolismMAPK1; PLK1; AKT2; PIK3CA; CDK8; PIK3CB; PIK3C3; MAPK8; MAPK3; PRKCD;PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; DYRK1A; MAP2K2; PIP5K1A; PIK3R1;MAP2K1; PAK3; ATM; TTK; CSNK1A1; BRAF; SGK PDGF Signaling EIF2AK2; ELK1;ABL2; MAPK1; PIK3CA; FOS; PIK3CB; PIK3C3; MAPK8; CAV1; ABL1; MAPK3;KRAS; SRC; PIK3C2A; PDGFRB; RAF1; MAP2K2; JAK1; JAK2; PIK3R1; PDGFRA;STAT3; SPHK1; MAP2K1; MYC; JUN; CRKL; PRKCA; SRF; STAT1; SPHK2 VEGFSignaling ACTN4; ROCK1; KDR; FLT1; ROCK2; MAPK1; PGF; AKT2; PIK3CA;ARNT; PTK2; BCL2; PIK3CB; PIK3C3; BCL2L1; MAPK3; KRAS; HIF1A; NOS3;PIK3C2A; PXN; RAF1; MAP2K2; ELAVL1; AKT1; PIK3R1; MAP2K1; SFN; VEGFA;AKT3; FOXO1; PRKCA Natural Killer Cell PRKCE; RAC1; PRKCZ; MAPK1; RAC2;PTPN11; Signaling KIR2DL3; AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3;PRKD1; MAPK3; KRAS; PRKCD; PTPN6; PIK3C2A; LCK; RAF1; FYN; MAP2K2; PAK4;AKT1; PIK3R1; MAP2K1; PAK3; AKT3; VAV3; PRKCA Cell Cycle: G1/S HDAC4;SMAD3; SUV39H1; HDAC5; CDKN1B; BTRC; Checkpoint Regulation ATR; ABL1;E2F1; HDAC2; HDAC7A; RB1; HDAC11; HDAC9; CDK2; E2F2; HDAC3; TP53;CDKN1A; CCND1; E2F4; ATM; RBL2; SMAD4; CDKN2A; MYC; NRG1; GSK3B; RBL1;HDAC6 T Cell Receptor RAC1; ELK1; MAPK1; IKBKB; CBL; PIK3CA; FOS;Signaling NFKB2; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; RELA; PIK3C2A; BTK;LCK; RAF1; IKBKG, RELB; FYN; MAP2K2; PIK3R1; CHUK; MAP2K1; NFKB1; ITK;BCL10; JUN; VAV3 Death Receptor Signaling CRADD; HSPB1; BID; BIRC4;TBK1; IKBKB; FADD; FAS; NFKB2; BCL2; MAP3K14; MAPK8; RIPK1; CASP8; DAXX;TNFRSF10B; RELA; TRAF2; TNF; IKBKG; RELB; CASP9; CHUK; APAF1; NFKB1;CASP2; BIRC2; CASP3; BIRC3 FGF Signaling RAC1; FGFR1; MET; MAPKAPK2;MAPK1; PTPN11; AKT2; PIK3CA; CREB1; PIK3CB; PIK3C3; MAPK8; MAPK3;MAPK13; PTPN6; PIK3C2A; MAPK14; RAF1; AKT1; PIK3R1; STAT3; MAP2K1;FGFR4; CRKL; ATF4; AKT3; PRKCA; HGF GN-CSF Signaling LYN; ELK1; MAPK1;PTPN11; AKT2; PIK3CA; CAMK2A; STAT5B; PIK3CB; PIK3C3; GNB2L1; BCL2L1;MAPK3; ETS1; KRAS; RUNX1; PIM1; PIK3C2A; RAF1; MAP2K2; AKT1; JAK2;PIK3R1; STAT3; MAP2K1; CCND1; AKT3; STAT1 Amyotrophic Lateral BID; IGF1;RAC1; BIRC4; PGF; CAPNS1; CAPN2; Sclerosis Signaling PIK3CA; BCL2;PIK3CB; PIK3C3; BCL2L1; CAPN1; PIK3C2A; TP53; CASP9; PIK3R1; RAB5A;CASP1; APAF1; VEGFA; BIRC2; BAX; AKT3; CASP3; BIRC3 JAK/Stat SignalingPTPN1; MAPK1; PTPN11; AKT2; PIK3CA; STAT5B; PIK3CB; PIK3C3; MAPK3; KRAS;SOCS1; STAT5A; PTPN6; PIK3C2A; RAF1; CDKN1A; MAP2K2; JAK1; AKT1; JAK2;PIK3R1; STAT3; MAP2K1; FRAP1; AKT3; STAT1 Nicotinate and PRKCE; IRAK1;PRKAA2; EIF2AK2; GRK6; MAPK1; Nicotinamide Metabolism PLK1; AKT2; T2;CDK8; MAPK8; MAPK3; PRKCD; PRKAA1; PBEF1; MAPK9; CDK2; PIMI; DYRK1A;MAP2K2; MAP2K1; PAK3; NT5E; TTK; CSNK1A1; BRAF; SGK Chemokine SignalingCXCR4; ROCK2; MAPK1; PTK2; FOS; CFL1; GNAQ; CAMK2A; CXCL12; MAPK8;MAPK3; KRAS; MAPK13; RHOA; CCR3; SRC; PPP1CC; MAPK14; NOX1; RAF1;MAP2K2; MAP2K1; JUN; CCL2; PRKCA IL-2 Signaling ELK1; MAPK1; PTPN11;AKT2; PIK3CA; SYK; FOS; STAT5B; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS;SOCS1; STAT5A; PIK3C2A; LCK; RAF1; MAP2K2; JAK1; AKT1; PIK3R1; MAP2K1;JUN; AKT3 Synaptic Long Term PRKCE; IGF1; PRKCZ; PRDX6; LYN; MAPK1;GNAS; Depression PRKCI; GNAQ; PPP2R1A; IGF1R; PRKD1; MAPK3; KRAS; GRN;PRKCD; NOS3; NOS2A; PPP2CA; YWHAZ; RAF1; MAP2K2; PPP2R5C; MAP2K1; PRKCAEstrogen Receptor TAF4B; EP300; CARM1; PCAF; MAPK1; NCOR2; SignalingSMARCA4; MAPK3; NRIP1; KRAS; SRC; NR3C1; HDAC3; PPARGG1A; RBM9; NCOA3;RAF1; CREBBP; MAP2K2; NCOA2; MAP2K1; PRKDC; ESR1; ESR2 ProteinUbiquitination TRAF6; SMURF1; BIRC4; BRCA1; UCHL1; NEDD4; Pathway CBL;UBE2I; BTRC; HSPA5; USP7; USP10; FBXW7; USP9X; STUB1; USP22; B2M; BIRC2;PARK2; USP8; USP1; VHL; HSP90AA1; BIRC3 IL-10 Signaling TRAF6; CCR1;ELK1; IKBKB; SP1; FOS; NFKB2; MAP3K14; MAPK8; MAPK13; RELA; MAPK14; TNF;IKBKG; RELB; MAP3K7; JAK1; CHUK; STAT3; NFKB1; JUN; IL1R1; IL6 VDR/RXRActivation PRKCE; EP300; PRKCZ; RXRA; GADD45A; HES1; NCOR2; SP1; PRKCI;CDKN1B; PRKD1; PRKCD; RUNX2; KLF4; YY1; NCOA3; CDKN1A; NCOA2; SPP1;LRP5; CEBPB; FOXO1; PRKCA TGF-beta Signaling EP300; SMAD2; SMURF1;MAPK1; SMAD3; SMAD1; FOS; MAPK8; MAPK3; KRAS; MAPK9; RUNX2; SERPINE1;RAF1; MAP3K7; CREBBP; MAP2K2; MAP2K1; TGFBR1; SMAD4; JUN; SMAD5Toil-like Receptor IRAK1; EIF2AK2; MYD88; TRAF6; PPARA; ELK1; SignalingIKBKB; FOS; NFKB2; MAP3K14; MAPK8; MAPK13; RELA; TLR4; MAPK14; IKBKG;RELB; MAP3K7; CHUK; NFKB1; TLR2; JUN p38 MAPK Signaling HSPB1; IRAK1;TRAF6; MAPKAPK2; ELK1; FADD; FAS; CREB1; DDIT3; RPS6KA4; DAXX; MAPK13;TRAF2; MAPK14; TNF; MAP3K7; TGFBR1; MYC; ATF4; IL1R1; SRF; STAT1Neurotrophin/TRK NTRK2; MAPK1; PTPN11; PIK3CA; CREB1; FOS; SignalingPIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; PIK3C2A; RAF1; MAP2K2; AKT1; PIK3R1;PDPK1; MAP2K1; CDC42; JUN; ATF4 FXR/RXR Activation INS; PPARA; FASN;RXRA; AKT2; SDC1; MAPK8; APOB; MAPK10; PPARG; MTTP; MAPK9; PPARGC1A;TNF; CREBBP; AKT1; SREBF1; FGFR4; AKT3; FOXO1 Synaptic Long Term PRKCE;RAP1A; EP300; PRKCZ; MAPK1; CREB1; Potentiation PRKCI; GNAQ; CAMK2A;PRKD1; MAPK3; KRAS; PRKCD; PPP1CC; RAF1; CREBBP; MAP2K2; MAP2K1; ATF4;PRKCA Calcium Signaling RAP1A; EP300; HDAC4; MAPK1; HDAC5; CREB1;CAMK2A; MYH9; MAPK3; HDAC2; HDAC7A; HDAC11; HDAC9; HDAC3; CREBBP; CALR;CAMKK2; ATF4; HDAC6 EGF Signaling ELK1; MAPK1; EGFR; PIK3CA; FOS;PIK3CB; PIK3C3; MAPK8; MAPK3; PIK3C2A; RAF1; JAK1; PIK3R1; STAT3;MAP2K1; JUN; PRKCA; SRF; STAT1 Hypoxia Signaling in the EDN1; PTEN;EP300; NQO1; UBE2I; CREB1; ARNT; Cardiovascular System HIF1A; SLC2A4;NOS3; TP53; LDHA; AKT1; ATM; VEGFA; JUN; ATF4; VHL; HSP90AA1 LPS/IL-1Mediated IRAK1; MYD88; TRAF6; PPARA; RXRA; ABCA1; Inhibition MAPK8;ALDH1A1; GSTP1; MAPK9; ABCB1; TRAF2; of RXR Function TLR4; TNF; MAP3K7;NR1H2; SREBF1; JUN; IL1R1 LXR/RXR Activation FASN; RXRA; NCOR2; ABCA1;NFKB2; IRF3; RELA; NOS2A; TLR4; TNF; RELB; LDLR; NR1H2; NFKB1; SREBF1;IL1R1; CCL2; IL6; MMP9 Amyloid Processing PRKCE; CSNK1E; MAPK1; CAPNS1;AKT2; CAPN2; CAPN1; MAPK3; MAPK13; MAPT; MAPK14; AKT1; PSEN1; CSNK1A1;GSK3B; AKT3; APP IL-4 Signaling AKT2; PIK3CA; PIK3CB; PIK3C3; IRS1;KRAS; SOCS1; PTPN6; NR3C1; PIK3C2A; JAK1; AKT1; JAK2; PIK3R1; FRAP1;AKT3; RPS6KB1 Cell Cycle: G2/M DNA EP300; PCAF; BRCA1; GADD45A; PLK1;BTRC; Damage Checkpoint CHEK1; ATR; CHEK2; YWHAZ; TP53; CDKN1A;Regulation PRKDC; ATM; SFN; CDKN2A Nitric Oxide Signaling in KDR; FLT1;PGF; AKT2; PIK3CA; PIK3CB; PIK3C3; the Cardiovascular System CAV1;PRKCD; NOS3; PIK3C2A; AKT1; PIK3R1; VEGFA; AKT3; HSP90AA1 PurineMetabolism NME2; SMARCA4; MYH9; RRM2; ADAR; EIF2AK4; PKM2; ENTPD1;RAD51; RRM2B; TJP2; RAD51C; NT5E; POLD1; NME1 cAMP-mediated RAP1A;MAPK1; GNAS; CREB1; CAMK2A; MAPK3; Signaling SRC; RAF1; MAP2K2; STAT3;MAP2K1; BRAF; ATF4 Mitochondrial SOD2; MAPK8; CASP8; MAPK10; MAPK9;CASP9; Dysfunction PARK7; PSEN1; PARK2; APP; CASP3 Notch Signaling HES1;JAG1; NUMB; NOTCH4; ADAM17; NOTCH2; PSEN1; NOTCH3; NOTCH1; DLL4Endoplasmic Reticulum HSPA5; MAPK8; XBP1; TRAF2; ATF6; CASP9; ATF4;Stress Pathway EIF2AK3; CASP3 Pyrimidine Metabolism NME2; AICDA; RRM2;EIF2AK4; ENTPD1; RRM2B; NT5E; POLD1; NME1 Parkinson's Signaling UCHL1;MAPK8; MAPK13; MAPK14; CASP9; PARK7; PARK2; CASP3 Cardiac & Beta GNAS;GNAQ; PPP2R1A; GNB2L1; PPP2CA; PPP1CC; Adrenergic Signaling PPP2R5CGlycolysis/Gluconeogenesis HK2; GCK; GPI; ALDH1A1; PKM2; LDHA; HK1Interferon Signaling IRF1; SOCS1; JAK1; JAK2; IFITM1; STAT1; IFIT3 SonicHedgehog ARRB2; SMO; GLI2; DYRK1A; GLI1; GSK39; DYRK1B SignalingGlycerophospholipid PLD1; GRN; GPAM; YWHAZ; SPHK1; SPHK2 MetabolismPhospholipid PRDX6; PLD1; GRN; YWHAZ; SPHK1; SPHK2 DegradationTryptophan Metabolism SIAH2; PRMT5; NEDD4; ALDH1A1; CYP1B1; SIAH1 LysineDegradation SUV39H1; EHMT2; NSD1; SETD7; PPP2R5C Nucleotide ExcisionERCC5; ERCC4; XPA; XPC; ERCC1 Repair Pathway Starch and Sucrose UCHL1;HK2; GCK; GPI; HK1 Metabolism Aminosugars Metabolism NQO1; HK2; GCK; HK1Arachidonic Acid PRDX6; GRN; YWHAZ; CYP1B1 Metabolism Circadian RhythmCSNK1E; CREB1; ATF4; NR1D1 Signaling Coagulation System BDKRB1; F2R;SERPINE1; F3 Dopamine Receptor PPP2R1A; PPP2CA; PPP1CC; PPP2R5CSignaling Glutathione Metabolism IDH2; GSTP1; ANPEP; IDH1 GlycerolipidMetabolism ALDH1A1; GPAM; SPHK1; SPHK2 Linoleic Acid PRDX6; GRN; YWHAZ;CYP1B1 Metabolism Methionine Metabolism DNMT1; DNMT3B; AHCY; DNMT3APyruvate Metabolism GLO1; ALDH1A1; PKM2; LDHA Arginine and ProlineALDH1A1; NOS3; NOS2A Metabolism Eicosanoid Signaling PRDX6; GRN; YWHAZFructose and Mannose HK2; GCK; HK1 Metabolism Galactose Metabolism HK2;GCK; HK1 Stilbene, Coumarine and PRDX6; PRDX1; TYR Lignin BiosynthesisAntigen Presentation CALR; B2M Pathway Biosynthesis of Steroids NQO1;DHCR7 Butanoate Metabolism ALDH1A1; NLGN1 Citrate Cycle IDH2; IDH1 FattyAcid Metabolism ALDH1A1; CYP1B1 Glycerophospholipid PRDX6; CHKAMetabolism Histidine Metabolism PRMT5; ALDH1A1 Inositol MetabolismERO1L; APEX1 Metabolism of GSTP1; CYP1B1 Xenobiotics by Cytochrome p450Methane Metabolism PRDX6; PRDX1 Phenylalanine PRDX6; PRDX1 MetabolismPropanoate Metabolism ALDH1A1; LDHA Selenoamino Acid PRMT5; AHCYMetabolism Sphingolipid Metabolism SPHK1; SPHK2 Aminophosphonate PRMT5Metabolism Androgen and Estrogen PRMT5 Metabolism Ascorbate and AldarateALDH1A1 Metabolism Bile Acid Biosynthesis ALDH1A1 Cysteine MetabolismLDHA Fatty Acid Biosynthesis FASN Glutamate Receptor GNB2L1 SignalingNRF2-mediated PRDX1 Oxidative Stress Response Pentose Phosphate GPIPathway Pentose and Glucuronate UCHL1 Interconversions RetinolMetabolism ALDH1A1 Riboflavin Metabolism TYR Tyrosine Metabolism PRMT5,TYR Ubiquinone Biosynthesis PRMT5 Valine, Leucine and ALDH1A1 IsoleucineDegradation Glycine, Serine and CHKA Threonine Metabolism LysineDegradation ALDH1A1 Pain/Taste TRPM5; TRPA1 Pain TRPM7; TRPC5; TRPC6;TRPC1; Cnr1; cnr2; Grk2; Trpa1; Pomc; Cgrp; Crf; Pka; Era; Nr2b; TRPM5;Prkaca; Prkacb; Prkar1a; Prkar2a Mitochondrial Function AIF; CytC; SMAC(Diablo); Aifm-1; Aifm-2 Developmental BMP-4; Chordin (Chrd); Noggin(Nog); WNT (Wnt2; Neurology Wnt2b, Wnt3a, Wnt4; Wnt5a; Wnt6; Wnt7b;Wnt8b; Wnt9a; Wnt9b; Wnt10a; Wnt10b, Wnt16); beta-catenin; Dkk-1;Frizzled related proteins; Otx-2; Gbx2; FGF-8; Reelin; Dab1; unc-86(Pou4f1 or Brn3a); Numb; Reln

Embodiments of the invention also relate to methods and compositionsrelated to knocking out genes, amplifying genes and repairing particularmutations associated with DNA repeat instability and neurologicaldisorders (Robert D. Wells, Tetsuo Ashizawa, Genetic Instabilities andNeurological Diseases, Second Edition, Academic Press, Oct. 13,2011—Medical). Specific aspects of tandem repeat sequences have beenfound to be responsible for more than twenty human diseases (Newinsights into repeat instability: role of RNA•DNA hybrids. McIvor E I,Polak U, Napierala M. RNA Biol. 2010 September-October; 7(5):551-8). TheCRISPR-Cas system may be harnessed to correct these defects of genomicinstability.

A further aspect of the invention relates to utilizing the CRISPR-Cassystem for correcting defects in the EMP2A and EMP2B genes that havebeen identified to be associated with Lafora disease. Lafora disease isan autosomal recessive condition which is characterized by progressivemyoclonus epilepsy which may start as epileptic seizures in adolescence.A few cases of the disease may be caused by mutations in genes yet to beidentified. The disease causes seizures, muscle spasms, difficultywalking, dementia, and eventually death. There is currently no therapythat has proven effective against disease progression. Other geneticabnormalities associated with epilepsy may also be targeted by theCRISPR-Cas system and the underlying genetics is further described inGenetics of Epilepsy and Genetic Epilepsies, edited by GiulianoAvanzini, Jeffrey L. Noebels, Mariani Foundation PaediatricNeurology:20; 2009).

In yet another aspect of the invention, the CRISPR-Cas system may beused to correct ocular defects that arise from several genetic mutationsfurther described in Genetic Diseases of the Eye, Second Edition, editedby Elias I. Traboulsi, Oxford University Press, 2012.

Several further aspects of the invention relate to correcting defectsassociated with a wide range of genetic diseases which are furtherdescribed on the website of the National Institutes of Health under thetopic subsection Genetic Disorders (website athealth.nih.gov/topic/GeneticDisorders). The genetic brain diseases mayinclude but are not limited to Adrenoleukodystrophy. Agenesis of theCorpus Callosum, Aicardi Syndrome, Alpers' Disease, Alzheimer's Disease,Barth Syndrome, Batten Disease, CADASIL, Cerebellar Degeneration,Fabry's Disease, Gerstmann-Straussler-Scheinker Disease, Huntington'sDisease and other Triplet Repeat Disorders, Leigh's Disease, Lesch-NyhanSyndrome, Menkes Disease, Mitochondrial Myopathies and NINDSColpocephaly. These diseases are further described on the website of theNational Institutes of Health under the subsection Genetic BrainDisorders.

In some embodiments, the condition may be neoplasia. In someembodiments, where the condition is neoplasia, the genes to be targetedare any of those listed in Table A (in this case PTEN asn so forth). Insome embodiments, the condition may be Age-related Macular Degeneration.In some embodiments, the condition may be a Schizophrenic Disorder. Insome embodiments, the condition may be a Trinucleotide Repeat Disorder.In some embodiments, the condition may be Fragile X Syndrome. In someembodiments, the condition may be a Secretase Related Disorder. In someembodiments, the condition may be a Prion-related disorder. In someembodiments, the condition may be ALS. In some embodiments, thecondition may be a drug addiction. In some embodiments, the conditionmay be Autism. In some embodiments, the condition may be Alzheimer'sDisease. In some embodiments, the condition may be inflammation. In someembodiments, the condition may be Parkinson's Disease.

Examples of proteins associated with Parkinson's disease include but arenot limited to α-synuclein, DJ-1, LRRK2, PINK1, Parkin, UCHL1,Synphilin-1, and NURR1.

Examples of addiction-related proteins may include ABAT for example.

Examples of inflammation-related proteins may include the monocytechemoattractant protein-1 (MCP1) encoded by the Ccr2 gene, the C-Cchemokine receptor type 5 (CCR5) encoded by the Ccr5 gene, the IgGreceptor IIB (FCGR2b, also termed CD32) encoded by the Fcgr2b gene, orthe Fc epsilon R1g (FCER1g) protein encoded by the Fcer1g gene, forexample.

Examples of cardiovascular diseases associated proteins may include IL1B(interleukin 1, beta), XDH (xanthine dehydrogenase), TP53 (tumor proteinp53), PTGIS (prostaglandin I2 (prostacyclin) synthase), MB (myoglobin),IL4 (interleukin 4), ANGPT1 (angiopoietin 1), ABCG8 (ATP-bindingcassette, sub-family G (WHITE), member 8), or CTSK (cathepsin K), forexample.

Examples of Alzheimer's disease associated proteins may include the verylow density lipoprotein receptor protein (VLDLR) encoded by the VLDLRgene, the ubiquitin-like modifier activating enzyme 1 (UBA1) encoded bythe UBA1 gene, or the NEDD8-activating enzyme E1 catalytic subunitprotein (UBE1C) encoded by the UBA3 gene, for example.

Examples of proteins associated Autism Spectrum Disorder may include thebenzodiazapine receptor (peripheral) associated protein 1 (BZRAP1)encoded by the BZRAP1 gene, the AF4/FMR2 family member 2 protein (AFF2)encoded by the AFF2 gene (also termed MFR2), the fragile X mentalretardation autosomal homolog 1 protein (FXR1) encoded by the FXR1 gene,or the fragile X mental retardation autosomal homolog 2 protein (FXR2)encoded by the FXR2 gene, for example.

Examples of proteins associated Macular Degeneration may include theATP-binding cassette, sub-family A (ABC1) member 4 protein (ABCA4)encoded by the ABCR gene, the apolipoprotein E protein (APOE) encoded bythe APOE gene, or the chemokine (C-C motif) Ligand 2 protein (CCL2)encoded by the CCL2 gene, for example.

Examples of proteins associated Schizophrenia may include NRG1, ErbB4,CPLX1, TPH1, TPH2, NRXN1, GSK3A, BDNF, DISC1, GSK3B, and combinationsthereof.

Examples of proteins involved in tumor suppression may include ATM(ataxia telangiectasia mutated), ATR (ataxia telangiectasia and Rad3related), EGFR (epidermal growth factor receptor), ERBB2 (v-erb-b2erythroblastic leukemia viral oncogene homolog 2), ERBB3 (v-erb-b2erythroblastic leukemia viral oncogene homolog 3), ERBB4 (v-crb-b2erythroblastic leukemia viral oncogene homolog 4), Notch 1, Notch2,Notch 3, or Notch 4, for example.

Examples of proteins associated with a secretase disorder may includePSENEN (presenilin enhancer 2 homolog (C. elegans)), CTSB (cathepsin B),PSEN (presenilin 1), APP (amyloid beta (A4) precursor protein), APH1B(anterior pharynx defective 1 homolog B (C. elegans)), PSEN2 (presenilin2 (Alzheimer disease 4)), or BACE1 (beta-site APP-cleaving enzyme 1),for example.

Examples of proteins associated with Amyotrophic Lateral Sclerosis mayinclude SOD1 (superoxide dismutase 1), ALS2 (amyotrophic lateralsclerosis 2), FUS (fused in sarcoma), TARDBP (TAR DNA binding protein),VAGFA (vascular endothelial growth factor A), VAGFB (vascularendothelial growth factor B), and VAGFC (vascular endothelial growthfactor C), and any combination thereof.

Examples of proteins associated with prion diseases may include SOD1(superoxide dismutase 1), ALS2 (amyotrophic lateral sclerosis 2), FUS(fused in sarcoma), TARDBP (TAR DNA binding protein), VAGFA (vascularendothelial growth factor A), VAGFB (vascular endothelial growth factorB), and VAGFC (vascular endothelial growth factor C), and anycombination thereof.

Examples of proteins related to neurodegenerative conditions in priondisorders may include A2M (Alpha-2-Macroglobulin), AATF (Apoptosisantagonizing transcription factor), ACPP (Acid phosphatase prostate),ACTA2 (Actin alpha 2 smooth muscle aorta), ADAM22 (ADAM metallopeptidasedomain), ADORA3 (Adenosine A3 receptor), or ADRA1D (Alpha-1D adrenergicreceptor for Alpha-1D adrenoreceptor), for example.

Examples of proteins associated with Immunodeficiency may include A2M[alpha-2-macroglobulin]; AANAT [arylalkylamine N-acetyltransferase];ABCA1 [ATP-binding cassette, sub-family A (ABC1), member 1]; ABCA2[ATP-binding cassette, sub-family A (ABC1), member 2]; or ABCA3[ATP-binding cassette, sub-family A (ABC1), member 3]; for example.

Examples of proteins associated with Trinucleotide Repeat Disordersinclude AR (androgen receptor), FMR1 (fragile X mental retardation 1),HTT (huntingtin), or DMPK (dystrophia myotonica-protein kinase), FXN(frataxin), ATXN2 (ataxin 2), for example.

Examples of proteins associated with Neurotransmission Disorders includeSST (somatostatin), NOS1 (nitric oxide synthase 1 (neuronal)), ADRA2A(adrenergic, alpha-2A-, receptor), ADRA2C (adrenergic, alpha-2C-,receptor), TACR1 (tachykinin receptor 1), or HTR2c (5-hydroxytryptamine(serotonin) receptor 2C), for example.

Examples of neurodevelopmental-associated sequences include A2BPI[ataxin 2-binding protein 1], AADAT [aminoadipate aminotransferase],AANAT [arylalkylamine N-acetyltransferase], ABAT [4-aminobutyrateaminotransferase], ABCA1 [ATP-binding cassette, sub-family A (ABC1),member 1], or ABCA13 [ATP-binding cassette, sub-family A (ABC1), member13], for example.

Further examples of preferred conditions treatable with the presentsystem include may be selected from: Aicardi-Goutières Syndrome;Alexander Disease; Allan-Herndon-Dudley Syndrome; POLG-RelatedDisorders; Alpha-Mannosidosis (Type II and III); Alström Syndrome;Angelman; Syndrome; Ataxia-Telangiectasia; NeuronalCeroid-Lipofuscinoses; Beta-Thalassemia; Bilateral Optic Atrophy and(Infantile) Optic Atrophy Type 1; Retinoblastoma (bilateral); CanavanDisease; Cerebrooculofacioskeletal Syndrome 1 [COFS1]; CerebrotendinousXanthomatosis; Cornelia de Lange Syndrome; MAPT-Related Disorders;Genetic Prion Diseases; Dravet Syndrome; Early-Onset Familial AlzheimerDisease; Friedreich Ataxia [FRDA]; Fryns Syndrome; Fucosidosis; FukuyamaCongenital Muscular Dystrophy; Galactosialidosis; Gaucher Disease;Organic Acidemias; Hemophagocytic Lymphohistiocytosis;Hutchinson-Gilford Progeria Syndrome; Mucolipidosis II; Infantile FreeSialic Acid Storage Disease; PLA2G6-Associated Neurodegeneration;Jervell and Lange-Nielsen Syndrome; Junctional Epidermolysis Bullosa;Huntington Disease; Krabbe Disease (Infantile); MitochondrialDNA-Associated Leigh Syndrome and NARP; Lesch-Nyhan Syndrome;LISI-Associated Lissencephaly; Lowe Syndrome; Maple Syrup Urine Disease;MECP2 Duplication Syndrome; ATP7A-Related Copper Transport Disorders;LAMA2-Related Muscular Dystrophy; Arylsulfatase A Deficiency;Mucopolysaccharidosis Types I, II or III; Peroxisome BiogenesisDisorders, Zellweger Syndrome Spectrum; Neurodegeneration with BrainIron Accumulation Disorders; Acid Sphingomyelinase Deficiency;Niemann-Pick Disease Type C; Glycine Encephalopathy; ARX-RelatedDisorders; Urea Cycle Disorders; COL1A1/2-Related OsteogenesisImperfecta; Mitochondrial DNA Deletion Syndromes; PLP1-RelatedDisorders; Perry Syndrome; Phelan-McDermid Syndrome; Glycogen StorageDisease Type II (Pompe Disease) (Infantile); MAPT-Related Disorders;MECP2-Related Disorders; Rhizomelic Chondrodysplasia Punctata Type 1;Roberts Syndrome; Sandhoff Disease; Schindler Disease—Type 1; AdenosineDeaminase Deficiency; Smith-Lemli-Opitz Syndrome; Spinal MuscularAtrophy; Infantile-Onset Spinocerebellar Ataxia; Hexosaminidase ADeficiency; Thanatophoric Dysplasia Type 1; Collagen Type VI-RelatedDisorders; Usher Syndrome Type I; Congenital Muscular Dystrophy;Wolf-Hirschhorn Syndrome; Lysosomal Acid Lipase Deficiency; andXeroderma Pigmentosum.

As will be apparent, it is envisaged that the present system can be usedto target any polynucleotide sequence of interest. Some examples ofconditions or diseases that might be usefully treated using the presentsystem are included in the Tables above and examples of genes currentlyassociated with those conditions are also provided there. However, thegenes exemplified are not exhaustive.

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. The present examples, along with the methodsdescribed herein are presently representative of preferred embodiments,are exemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses which are encompassed withinthe spirit of the invention as defined by the scope of the claims willoccur to those skilled in the art.

Example 1 CRISPR Complex Activity in the Nucleus of a Eukaryotic Cell

An example type II CRISPR system is the type II CRISPR locus fromStreptococcus pyogenes SF370, which contains a cluster of four genesCas9, Cas1, Cas2, and Csn1, as well as two non-coding RNA elements,tracrRNA and a characteristic array of repetitive sequences (directrepeats) interspaced by short stretches of non-repetitive sequences(spacers, about 30 bp each). In this system, targeted DNA double-strandbreak (DSB) is generated in four sequential steps (FIG. 2A). First, twonon-coding RNAs, the pre-crRNA array and tracrRNA, are transcribed fromthe CRISPR locus. Second, tracrRNA hybridizes to the direct repeats ofpre-crRNA, which is then processed into mature crRNAs containingindividual spacer sequences. Third, the mature crRNA:tracrRNA complexdirects Cas9 to the DNA target consisting of the protospacer and thecorresponding PAM via heteroduplex formation between the spacer regionof the crRNA and the protospacer DNA. Finally, Cas9 mediates cleavage oftarget DNA upstream of PAM to create a DSB within the protospacer (FIG.2A). This example describes an example process for adapting thisRNA-programmable nuclease system to direct CRISPR complex activity inthe nuclei of eukaryotic cells.

Cell Culture and Transfection

Human embryonic kidney (HEK) cell line HEK 293FT (Life Technologies) wasmaintained in Dulbecco's modified Eagle's Medium (DMEM) supplementedwith 10% fetal bovine serum (HyClone), 2 mM GlutaMAX (LifeTechnologies), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37° C.with 5% CO₂ incubation. Mouse neuro2A (N2A) cell line (ATCC) wasmaintained with DMEM supplemented with 5% fetal bovine serum (HyClone),2 mM GlutaMAX (Life Technologies), 100 U/mL penicillin, and 100 μg/mLstreptomycin at 37° C. with 5% CO₂.

HEK 293FT or N2A cells were seeded into 24-well plates (Corning) one dayprior to transfection at a density of 200,000 cells per well. Cells weretransfected using Lipofectamine 2000 (Life Technologies) following themanufacturer's recommended protocol. For each well of a 24-well plate atotal of 800 ng of plasmids were used.

Surveyor Assay and Sequencing Analysis for Genome Modification

HEK 293FT or N2A cells were transfected with plasmid DNA as describedabove. After transfection, the cells were incubated at 37° C. for 72hours before genomic DNA extraction. Genomic DNA was extracted using theQuickExtract DNA extraction kit (Epicentre) following the manufacturer'sprotocol. Briefly, cells were resuspended in QuickExtract solution andincubated at 65° C. for 15 minutes and 98° C. for 10 minutes. Extractedgenomic DNA was immediately processed or stored at −20° C.

The genomic region surrounding a CRISPR target site for each gene wasPCR amplified, and products were purified using QiaQuick Spin Column(Qiagen) following manufacturer's protocol. A total of 400 ng of thepurified PCR products were mixed with 2 μl 10×Taq polymerase PCR buffer(Enzymatics) and ultrapure water to a final volume of 20 μl, andsubjected to a re-annealing process to enable heteroduplex formation:95° C. for 10 min, 95° C. to 85° C. ramping at −2° C./s, 85° C. to 25°C. at −0.25° C./s, and 25° C. hold for 1 minute. After re-annealing,products were treated with Surveyor nuclease and Surveyor enhancer S(Transgenomics) following the manufacturer's recommended protocol, andanalyzed on 4-20% Novex TBE poly-acrylamide gels (Life Technologies).Gels were stained with SYBR Gold DNA stain (Life Technologies) for 30minutes and imaged with a Gel Doc gel imaging system (Bio-rad).Quantification was based on relative band intensities, as a measure ofthe fraction of cleaved DNA. FIG. 7 provides a schematic illustration ofthis Surveyor assay.

Restriction fragment length polymorphism assay for detection ofhomologous recombination.

HEK 293FT and N2A cells were transfected with plasmid DNA, and incubatedat 37° C. for 72 hours before genomic DNA extraction as described above.The target genomic region was PCR amplified using primers outside thehomology arms of the homologous recombination (HR) template. PCRproducts were separated on a 1% agarose gel and extracted with MinEluteGelExtraction Kit (Qiagen). Purified products were digested with HindIII(Fermentas) and analyzed on a 6% Novex TBE poly-acrylamide gel (LifeTechnologies).

RNA Secondary Structure Prediction and Analysis

RNA secondary structure prediction was performed using the onlinewebserver RNAfold developed at Institute for Theoretical Chemistry atthe University of Vienna, using the centroid structure predictionalgorithm (see e.g. A. R. Gruber et al., 2008, Cell 106(1): 23-24; andPA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62).

RNA Purification

HEK 293FT cells were maintained and transfected as stated above. Cellswere harvested by trypsinization followed by washing in phosphatebuffered saline (PBS). Total cell RNA was extracted with TRI reagent(Sigma) following manufacturer's protocol. Extracted total RNA wasquantified using Naonodrop (Thermo Scientific) and normalized to sameconcentration.

Northern Blot Analysis of crRNA and tracrRNA Expression in MammalianCells

RNAs were mixed with equal volumes of 2× loading buffer (Ambion), heatedto 95° C. for 5 min, chilled on ice for 1 min, and then loaded onto 8%denaturing polyacrylamide gels (SequaGel, National Diagnostics) afterpre-running the gel for at least 30 minutes. The samples wereelectrophoresed for 1.5 hours at 40 W limit. Afterwards, the RNA wastransferred to Hybond N+ membrane (GE Healthcare) at 300 mA in asemi-dry transfer apparatus (Bio-rad) at room temperature for 1.5 hours.The RNA was crosslinked to the membrane using autocrosslink button onStratagene UV Crosslinker the Stratalinker (Stratagene). The membranewas pre-hybridized in ULTRAhyb-Oligo Hybridization Buffer (Ambion) for30 min with rotation at 42° C., and probes were then added andhybridized overnight. Probes were ordered from IDT and labeled with[gamma-³²P] ATP (Perkin Elmer) with T4 polynucleotide kinase (NewEngland Biolabs). The membrane was washed once with pre-warmed (42° C.)2×SSC, 0.5% SDS for 1 min followed by two 30 minute washes at 42° C. Themembrane was exposed to a phosphor screen for one hour or overnight atroom temperature and then scanned with a phosphorimager (Typhoon).

Bacterial CRISPR System Construction and Evaluation

CRISPR locus elements, including tracrRNA, Cas9, and leader were PCRamplified from Streptococcus pyogenes SF370 genomic DNA with flankinghomology arms for Gibson Assembly. Two Bsa type IIS sites wereintroduced in between two direct repeats to facilitate easy insertion ofspacers (FIG. 8). PCR products were cloned into EcoRV-digested pACYC184downstream of the tet promoter using Gibson Assembly Master Mix (NEB).Other endogenous CRISPR system elements were omitted, with the exceptionof the last 50 bp of Csn2. Oligos (Integrated DNA Technology) encodingspacers with complimentary overhangs were cloned into the BsaI-digestedvector pDC000 (NEB) and then ligated with T7 ligase (Enzymatics) togenerate pCRISPR plasmids. Challenge plasmids containing spacers withPAM

expression in mammalian cells (expression constructs illustrated in FIG.6A, with functionality as determined by results of the Surveyor assayshown in FIG. 6B). Transcription start sites are marked as +1, andtranscription terminator and the sequence probed by northern blot arealso indicated. Expression of processed tracrRNA was also confirmed byNorthern blot. FIG. 6C shows results of a Northern blot analysis oftotal RNA extracted from 293FT cells transfected with U6 expressionconstructs carrying long or short tracrRNA, as well as SpCas9 andDR-EMX1(1)-DR. Left and right panels are from 293FT cells transfectedwithout or with SpRNase III, respectively. U6 indicate loading controlblotted with a probe targeting human U6 snRNA. Transfection of the shorttracrRNA expression construct led to abundant levels of the processedform of tracrRNA (˜75 bp). Very low amounts of long tracrRNA aredetected on the Northern blot.

To promote precise transcriptional initiation, the RNA polymeraseIII-based U6 promoter was selected to drive the expression of tracrRNA(FIG. 2C). Similarly, a U6 promoter-based construct was developed toexpress a pre-crRNA array consisting of a single spacer flanked by twodirect repeats (DRs, also encompassed by the term “tracr-matesequences”; FIG. 2C). The initial spacer was designed to target a33-base-pair (bp) target site (30-bp protospacer plus a 3-bp CRISPRmotif (PAM) sequence satisfying the NGG recognition motif of Cas9) inthe human EMX1 locus (FIG. 2C), a key gene in the development of thecerebral cortex.

To test whether heterologous expression of the CRISPR system (SpCas9,SpRNase III, tracrRNA, and pre-crRNA) in mammalian cells can achievetargeted cleavage of mammalian chromosomes, HEK 293FT cells weretransfected with combinations of CRISPR components. Since DSBs inmammalian nuclei are partially repaired by the non-homologous endjoining (NHEJ) pathway, which leads to the formation of indels, theSurveyor assay was used to detect potential cleavage activity at thetarget EMX1 locus (FIG. 7) (see e.g. Guschin et al., 2010, Methods MolBiol 649: 247). Co-transfection of all four CRISPR components was ableto induce up to 5.0% cleavage in the protospacer (see FIG. 2D).Co-transfection of all CRISPR components minus SpRNase III also inducedup to 4.7% indel in the protospacer, suggesting that there may beendogenous mammalian RNases that are capable of assisting with crRNAmaturation, such as for example the related Dicer and Drosha enzymes.Removing any of the remaining three components abolished the genomecleavage activity of the CRISPR system (FIG. 2D). Sanger sequencing ofamplicons containing the target locus verified the cleavage activity: in43 sequenced clones, 5 mutated alleles (11.6%) were found. Similarexperiments using a variety of guide sequences produced indelpercentages as high as 29% (see FIGS. 3-6, 10, and 11). These resultsdefine a three-component system for efficient CRISPR-mediated genomemodification in mammalian cells. To optimize the cleavage efficiency,Applicants also tested whether different isoforms of tracrRNA affectedthe cleavage efficiency and found that, in this example system, only theshort (89-bp) transcript form was able to mediate cleavage of the humanEMX1 genomic locus (FIG. 6B).

FIG. 12 provides an additional Northern blot analysis of crRNAprocessing in mammalian cells. FIG. 12A illustrates a schematic showingthe expression vector for a single spacer flanked by two direct repeats(DR-EMX1(1)-DR). The 30 bp spacer targeting the human EMX1 locusprotospacer 1 (see FIG. 6) and the direct repeat sequences are shown inthe sequence beneath FIG. 12A. The line indicates the region whosereverse-complement sequence was used to generate Northern blot probesfor EMX1(1) crRNA detection. FIG. 12B shows a Northern blot analysis oftotal RNA extracted from 293FT cells transfected with U6 expressionconstructs carrying DR-EMX1(1)-DR. Left and right panels are from 293FTcells transfected without or with SpRNase III respectively.DR-EMX1(1)-DR was processed into mature crRNAs only in the presence ofSpCas9 and short tracrRNA and was not dependent on the presence ofSpRNase III. The mature crRNA detected from transfected 293FT total RNAis ˜33 bp and is shorter than the 39-42 bp mature crRNA from S.pyogenes. These results demonstrate that a CRISPR system can betransplanted into eukaryotic cells and reprogrammed to facilitatecleavage of endogenous mammalian target polynucleotides.

FIG. 2 illustrates the bacterial CRISPR system described in thisexample. FIG. 2A illustrates a schematic showing the CRISPR locus 1 fromStreptococcus pyogenes SF370 and a proposed mechanism of CRISPR-mediatedDNA cleavage by this system. Mature crRNA processed from the directrepeat-spacer array directs Cas9 to genomic targets consisting ofcomplimentary protospacers and a protospacer-adjacent motif (PAM). Upontarget-spacer base pairing, Cas9 mediates a double-strand break in thetarget DNA. FIG. 2B illustrates engineering of S. pyogenes Cas9 (SpCas9)and RNase 111 (SpRNase III) with nuclear localization signals (NLSs) toenable import into the mammalian nucleus. FIG. 2C illustrates mammalianexpression of SpCas9 and SpRNase III driven by the constitutive EF1apromoter and tracrRNA and pre-crRNA array (DR-Spacer-DR) driven by theRNA Pol3 promoter U6 to promote precise transcription initiation andtermination. A protospacer from the human EMX1 locus with a satisfactoryPAM sequence is used as the spacer in the pre-crRNA array. FIG. 2Dillustrates surveyor nuclease assay for SpCas9-mediated minor insertionsand deletions. SpCas9 was expressed with and without SpRNase III,tracrRNA, and a pre-crRNA array carrying the EMX1-target spacer. FIG. 2Eillustrates a schematic representation of base pairing between targetlocus and EMAX1-targeting crRNA, as well as an example chromatogramshowing a micro deletion adjacent to the SpCas9 cleavage site. FIG. 2Fillustrates mutated alleles identified from sequencing analysis of 43clonal amplicons showing a variety of micro insertions and deletions.Dashes indicate deleted bases, and non-aligned or mismatched basesindicate insertions or mutations. Scale bar=10 μm.

To further simplify the three-component system, a chimericcrRNA-tracrRNA hybrid design was adapted, where a mature crRNA(comprising a guide sequence) may be fused to a partial tracrRNA via astem-loop to mimic the natural crRNA:tracrRNA duplex. To increaseco-delivery efficiency, a bicistronic expression vector was created todrive co-expression of a chimeric RNA and SpCas9 in transfected cells.In parallel, the bicistronic vectors were used to express a pre-crRNA(DR-guide sequence-DR) with SpCas9, to induce processing into crRNA witha separately expressed tracrRNA (compare FIG. 11B top and bottom). FIG.8 provides schematic illustrations of bicistronic expression vectors forpre-crRNA array (FIG. 8A) or chimeric crRNA (represented by the shortline downstream of the guide sequence insertion site and upstream of theEF1α promoter in FIG. 8B) with hSpCas9, showing location of variouselements and the point of guide sequence insertion. The expandedsequence around the location of the guide sequence insertion site inFIG. 8B also shows a partial DR sequence (GTTTTAGAGCTA) (SEQ ID NO: 11)and a partial tracrRNA sequence (TAGCAAGTTAAAATAAGGCTAGTCCGTTTTTT) (SEQID NO: 12). Guide sequences can be inserted between BbsI sites usingannealed oligonucleotides. Sequence design for the oligonucleotides areshown below the schematic illustrations in FIG. 8, with appropriateligation adapters indicated. WPRE represents the Woodchuck hepatitisvirus post-transcriptional regulatory element. The efficiency ofchimeric RNA-mediated cleavage was tested by targeting the same EMX1locus described above. Using both Surveyor assay and Sanger sequencingof amplicons, Applicants confirmed that the chimeric RNA designfacilitates cleavage of human EMX1 locus with approximately a 4.7%modification rate (FIG. 3).

Generalizability of CRISPR-mediated cleavage in eukaryotic cells wastested by targeting additional genomic loci in both human and mousecells by designing chimeric RNA targeting multiple sites in the humanEMX1 and PVALB, as well as the mouse Th loci. FIG. 13 illustrates theselection of some additional targeted protospacers in human PVALB (FIG.13A) and mouse Th (FIG. 13B) loci. Schematics of the gene loci and thelocation of three protospacers within the last exon of each areprovided. The underlined sequences include 30 bp of protospacer sequenceand 3 bp at the 3′ end corresponding to the PAM sequences. Protospacerson the sense and anti-sense strands are indicated above and below theDNA sequences, respectively. A modification rate of 6.3% and 0.75% wasachieved for the human PVALB and mouse Th loci respectively,demonstrating the broad applicability of the CRISPR system in modifyingdifferent loci across multiple organisms (FIG. 5). While cleavage wasonly detected with one out of three spacers for each locus using thechimeric constructs, all target sequences were cleaved with efficiencyof indel production reaching 27% when using the co-expressed pre-crRNAarrangement (FIGS. 6 and 13).

FIG. 11 provides a further illustration that SpCas9 can be reprogrammedto target multiple genomic loci in mammalian cells. FIG. 11A provides aschematic of the human EMXNL locus showing the location of fiveprotospacers, indicated by the underlined sequences. FIG. 11B provides aschematic of the pre-crRNA/trcrRNA complex showing hybridization betweenthe direct repeat region of the pre-crRNA and tracrRNA (top), and aschematic of a chimeric RNA design comprising a 20 bp guide sequence,and tracr mate and tracr sequences consisting of partial direct repeatand tracrRNA sequences hybridized in a hairpin structure (bottom).Results of a Surveyor assay comparing the efficacy of Cas9-mediatedcleavage at five protospacers in the human EMX1 locus is illustrated inFIG. 11C. Each protospacer is targeted using either processedpre-crRNA/tracrRNA complex (crRNA) or chimeric RNA (chiRNA).

Since the secondary structure of RNA can be crucial for intermolecularinteractions, a structure prediction algorithm based on minimum freeenergy and Boltzmann-weighted structure ensemble was used to compare theputative secondary structure of all guide sequences used in the genometargeting experiment (see e.g. Gruber et al., 2008, Nucleic AcidsResearch, 36: W70). Analysis revealed that in most cases, the effectiveguide sequences in the chimeric crRNA context were substantially free ofsecondary structure motifs, whereas the ineffective guide sequences weremore likely to form internal secondary structures that could preventbase pairing with the target protospacer DNA. It is thus possible thatvariability in the spacer secondary structure might impact theefficiency of CRISPR-mediated interference when using a chimeric crRNA.

Further vector designs for SpCas9 are shown in FIG. 22, whichillustrates single expression vectors incorporating a U6 promoter linkedto an insertion site for a guide oligo, and a Cbh promoter linked toSpCas9 coding sequence. The vector shown in FIG. 22 b includes atracrRNA coding sequence linked to an H1 promoter.

In the bacterial assay, all spacers facilitated efficient CRISPRinterference (FIG. 3C). These results suggest that there may beadditional factors affecting the efficiency of CRISPR activity inmammalian cells.

To investigate the specificity of CRISPR-mediated cleavage, the effectof single-nucleotide mutations in the guide sequence on protospacercleavage in the mammalian genome was analyzed using a series ofEMX1-targeting chimeric crRNAs with single point mutations (FIG. 3A).FIG. 3B illustrates results of a Surveyor nuclease assay comparing thecleavage efficiency of Cas9 when paired with different mutant chimericRNAs. Single-base mismatch up to 12-bp 5′ of the PAM substantiallyabrogated genomic cleavage by SpCas9, whereas spacers with mutations atfarther upstream positions retained activity against the originalprotospacer target (FIG. 3B). In addition to the PAM, SpCas9 hassingle-base specificity within the last 12-bp of the spacer.Furthermore, CRISPR is able to mediate genomic cleavage as efficientlyas a pair of TALE nucleases (TALEN) targeting the same EMX1 protospacer.FIG. 3C provides a schematic showing the design of TALENs targetingEMX1, and FIG. 3D shows a Surveyor gel comparing the efficiency of TALENand Cas9 (n=3).

Having established a set of components for achieving CRISPR-mediatedgene editing in mammalian cells through the error-prone NHEJ mechanism,the ability of CRISPR to stimulate homologous recombination (HR), a highfidelity gene repair pathway for making precise edits in the genome, wastested. The wild type SpCas9 is able to mediate site-specific DSBs,which can be repaired through both NHEJ and HR. In addition, anaspartate-to-alanine substitution (D10A) in the RuvC I catalytic domainof SpCas9 was engineered to convert the nuclease into a nickase(SpCas9n; illustrated in FIG. 4A) (see e.g. Sapranausaks et al., 2011,Nucleic Acids Research, 39: 9275; Gasiunas et al., 2012, Proc. Natl.Acad. Sci. USA, 109:E2579), such that nicked genomic DNA undergoes thehigh-fidelity homology-directed repair (HDR). Surveyor assay confirmedthat SpCas9n does not generate indels at the EMX1 protospacer target. Asillustrated in FIG. 4B, co-expression of EMX1-targeting chimeric crRNAwith SpCas9 produced indels in the target site, whereas co-expressionwith SpCas9n did not (n=3). Moreover, sequencing of 327 amplicons didnot detect any indels induced by SpCas9n. The same locus was selected totest CRISPR-mediated HR by co-transfecting HEK 293FT cells with thechimeric RNA targeting EMX1, hSpCas9 or hSpCas9n, as well as a HRtemplate to introduce a pair of restriction sites (HindIII and NheI)near the protospacer. FIG. 4C provides a schematic illustration of theHR strategy, with relative locations of recombination points and primerannealing sequences (arrows). SpCas9 and SpCas9n indeed catalyzedintegration of the HR template into the EMX1 locus. PCR amplification ofthe target region followed by restriction digest with HindIII revealedcleavage products corresponding to expected fragment sizes (arrows inrestriction fragment length polymorphism gel analysis shown in FIG. 4D),with SpCas9 and SpCas9n mediating similar levels of HR efficiencies.Applicants further verified HR using Sanger sequencing of genomicamplicons (FIG. 4E). These results demonstrate the utility of CRISPR forfacilitating targeted gene insertion in the mammalian genome. Given the14-bp (12-bp from the spacer and 2-bp from the PAM) target specificityof the wild type SpCas9, the availability of a nickase can significantlyreduce the likelihood of off-target modifications, since single strandbreaks are not substrates for the error-prone NHEJ pathway.

Expression constructs mimicking the natural architecture of CRISPR lociwith arrayed spacers (FIG. 2A) were constructed to test the possibilityof multiplexed sequence targeting. Using a single CRISPR array encodinga pair of EMX1- and PVALB-targeting spacers, efficient cleavage at bothloci was detected (FIG. 4F, showing both a schematic design of the crRNAarray and a Surveyor blot showing efficient mediation of cleavage).Targeted deletion of larger genomic regions through concurrent DSBsusing spacers against two targets within EMX1 spaced by 119 bp was alsotested, and a 1.6% deletion efficacy (3 out of 182 amplicons; FIG. 4G)was detected. This demonstrates that the CRISPR system can mediatemultiplexed editing within a single genome.

Example 2 CRISPR System Modifications and Alternatives

The ability to use RNA to program sequence-specific DNA cleavage definesa new class of genome engineering tools for a variety of research andindustrial applications. Several aspects of the CRISPR system can befurther improved to increase the efficiency and versatility of CRISPRtargeting. Optimal Cas9 activity may depend on the availability of freeMg²⁺ at levels higher than that present in the mammalian nucleus (seee.g. Jinek et al., 2012, Science, 337:816), and the preference for anNGG motif immediately downstream of the protospacer restricts theability to target on average every 12-bp in the human genome (FIG. 9,evaluating both plus and minus strands of human chromosomal sequences).Some of these constraints can be overcome by exploring the diversity ofCRISPR loci across the microbial metagenome (see e.g. Makarova et al.,2011, Nat Rev Microbiol, 9:467). Other CRISPR loci may be transplantedinto the mammalian cellular milieu by a process similar to thatdescribed in Example 1. For example, FIG. 10 illustrates adaptation ofthe Type II CRISPR system from CRISPR 1 of Streptococcus thermophilusLMD-9 for heterologous expression in mammalian cells to achieveCRISPR-mediated genome editing. FIG. 10A provides a Schematicillustration of CRISPR 1 from S. thermophilus LMD-9. FIG. 10Billustrates the design of an expression system for the S. thermophilusCRISPR system. Human codon-optimized hStCas9 is expressed using aconstitutive EF1α promoter. Mature versions of tracrRNA and crRNA areexpressed using the U6 promoter to promote precise transcriptioninitiation. Sequences from the mature crRNA and tracrRNA areillustrated. A single base indicated by the lower case “a” in the crRNAsequence is used to remove the polyU sequence, which serves as a RNApolIII transcriptional terminator. FIG. 10C provides a schematic showingguide sequences targeting the human EMX1 locus. FIG. 10D shows theresults of hStCas9-mediated cleavage in the target locus using theSurveyor assay. RNA guide spacers 1 and 2 induced 14% and 6.4%,respectively. Statistical analysis of cleavage activity acrossbiological replica at these two protospacer sites is also provided inFIG. 5. FIG. 14 provides a schematic of additional protospacer andcorresponding PAM sequence targets of the S. thermophilus CRISPR systemin the human EMX1 locus. Two protospacer sequences are highlighted andtheir corresponding PAM sequences satisfying NNAGAAW motif are indicatedby underlining 3′ with respect to the corresponding highlightedsequence. Both protospacers target the anti-sense strand.

Example 3 Sample Target Sequence Selection Algorithm

A software program is designed to identify candidate CRISPR targetsequences on both strands of an input DNA sequence based on desiredguide sequence length and a CRISPR motif sequence (PAM) for a specifiedCRISPR enzyme. For example, target sites for Cas9 from S. pyogenes, withPAM sequences NGG, may be identified by searching for 5′-N_(x)-NGG-3′both on the input sequence and on the reverse-complement of the input.Likewise, target sites for Cas9 of S. thermophilus CRISPR1, with PAMsequence NNAGAAW, may be identified by searching for 5′-N_(x)-NNAGAAW-3′both on the input sequence and on the reverse-complement of the input.Likewise, target sites for Cas9 of S. thermophilus CRISPR3, with PAMsequence NGGNG, may be identified by searching for 5′-N_(x)-NGGNG-3′both on the input sequence and on the reverse-complement of the input.The value “x” in N_(x) may be fixed by the program or specified by theuser, such as 20.

Since multiple occurrences in the genome of the DNA target site may leadto nonspecific genome editing, after identifying all potential sites,the program filters out sequences based on the number of times theyappear in the relevant reference genome. For those CRISPR enzymes forwhich sequence specificity is determined by a ‘seed’ sequence, such asthe 11-12 bp 5′ from the PAM sequence, including the PAM sequenceitself, the filtering step may be based on the seed sequence. Thus, toavoid editing at additional genomic loci, results are filtered based onthe number of occurrences of the seed:PAM sequence in the relevantgenome. The user may be allowed to choose the length of the seedsequence. The user may also be allowed to specify the number ofoccurrences of the seed:PAM sequence in a genome for purposes of passingthe filter. The default is to screen for unique sequences. Filtrationlevel is altered by changing both the length of the seed sequence andthe number of occurrences of the sequence in the genome. The program mayin addition or alternatively provide the sequence of a guide sequencecomplementary to the reported target sequence(s) by providing thereverse complement of the identified target sequence(s). An examplevisualization of some target sites in the human genome is provided inFIG. 18.

Further details of methods and algorithms to optimize sequence selectioncan be found in U.S. application Ser. No. 61/064,798 (Broad ReferenceBI-2012/084); incorporated herein by reference.

Example 4 Evaluation of Multiple Chimeric crRNA-tracrRNA Hybrids

This example describes results obtained for chimeric RNAs (chiRNAs;comprising a guide sequence, a tracr mate sequence, and a tracr sequencein a single transcript) having tracr sequences that incorporatedifferent lengths of wild-type tracrRNA sequence. FIG. 16 a illustratesa schematic of a bicistronic expression vector for chimeric RNA andCas9. Cas9 is driven by the CBh promoter and the chimeric RNA is drivenby a U6 promoter. The chimeric guide RNA consists of a 20 bp guidesequence (Ns) joined to the tracr sequence (running from the first “U”of the lower strand to the end of the transcript), which is truncated atvarious positions as indicated. The guide and tracr sequences areseparated by the tracr-mate sequence GUUUUAGAGCUA (SEQ ID NO: 13)followed by the loop sequence GAAA. Results of SURVEYOR assays forCas9-mediated indels at the human EMX1 and PVALB loci are illustrated inFIGS. 16 b and 16 c, respectively. Arrows indicate the expected SURVEYORfragments. ChiRNAs are indicated by their “+n” designation, and crRNArefers to a hybrid RNA where guide and tracr sequences are expressed asseparate transcripts. Quantification of these results, performed intriplicate, are illustrated by histogram in FIGS. 17 a and 17 b,corresponding to FIGS. 16 b and 16 c, respectively (“N.D.” indicates noindels detected). Protospacer IDs and their corresponding genomictarget, protospacer sequence, PAM sequence, and strand location areprovided in Table D. Guide sequences were designed to be complementaryto the entire protospacer sequence in the case of separate transcriptsin the hybrid system, or only to the underlined portion in the case ofchimeric RNAs.

TABLE D SEQ protospacer genomic ID ID target protospacer sequence (5′to 3′) PAM NO: strand 1 EMX1 GGACATCGATGTCACCTCCAATGACTAGGG TGG 14 + 2EMX1 CATTGGAGGTGACATCGATGTCCTCCCCAT TGG 15 − 3 EMX1GGAAGGGCCTGAGTCCGAGCAGAAGAAGAA GGG 16 + 4 PVALBGGTGGCGAGAGGGGCCGAGATTGGGTGTTC AGG 17 + 5 PVALBATGCAGGAGGGTGGCGAGAGGGGCCGAGAT TGG 18 +

Further details to optimize guide sequences can be found in U.S.application Ser. No. 61/836,127 (Broad Reference BI-2013/004G) filedJun. 17, 2013; incorporated herein by reference.

Initially, three sites within the EMX1 locus in human HEK 293FT cellswere targeted. Genome modification efficiency of each chiRNA wasassessed using the SURVEYOR nuclease assay, which detects mutationsresulting from DNA double-strand breaks (DSBs) and their subsequentrepair by the non-homologous end joining (NHEJ) DNA damage repairpathway. Constructs designated chiRNA(+n) indicate that up to the +nnucleotide of wild-type tracrRNA is included in the chimeric RNAconstruct, with values of 48, 54, 67, and 85 used for n. Chimeric RNAscontaining longer fragments of wild-type tracrRNA (chiRNA(+67) andchiRNA(+85)) mediated DNA cleavage at all three EMX1 target sites, withchiRNA(+85) in particular demonstrating significantly higher levels ofDNA cleavage than the corresponding crRNA/tracrRNA hybrids thatexpressed guide and tracr sequences in separate transcripts (FIGS. 16 band 17 a). Two sites in the PVALB locus that yielded no detectablecleavage using the hybrid system (guide sequence and tracr sequenceexpressed as separate transcripts) were also targeted using chiRNAs.chiRNA(+67) and chiRNA(+85) were able to mediate significant cleavage atthe two PVALB protospacers (FIGS. 16 c and 17 b).

For all five targets in the EMX1 and PVALB loci, a consistent increasein genome modification efficiency with increasing tracr sequence lengthwas observed. Without wishing to be bound by any theory, the secondarystructure formed by the 3′ end of the tracrRNA may play a role inenhancing the rate of CRISPR complex formation.

Example 5 Cas9 Diversity

The CRISPR-Cas system is an adaptive immune mechanism against invadingexogenous DNA employed by diverse species across bacteria and archaea.The type II CRISPR-Cas9 system consists of a set of genes encodingproteins responsible for the “acquisition” of foreign DNA into theCRISPR locus, as well as a set of genes encoding the “execution” of theDNA cleavage mechanism; these include the DNA nuclease (Cas9), anon-coding transactivating cr-RNA (tracrRNA), and an array of foreignDNA-derived spacers flanked by direct repeats (crRNAs). Upon maturationby Cas9, the tracRNA and crRNA duplex guide the Cas9 nuclease to atarget DNA sequence specified by the spacer guide sequences, andmediates double-stranded breaks in the DNA near a short sequence motifin the target DNA that is required for cleavage and specific to eachCRISPR-Cas system. The type II CRISPR-Cas systems are found throughoutthe bacterial kingdom and highly diverse in Cas9 protein sequence andsize, tracrRNA and crRNA direct repeat sequence, genome organization ofthese elements, and the motif requirement for target cleavage. Onespecies may have multiple distinct CRISPR-Cas systems.

Applicants evaluated 207 putative Cas9s from bacterial speciesidentified based on sequence homology to known Cas9s and structuresorthologous to known subdomains, including the HNH endonuclease domainand the RuvC endonuclease domains [information from the Eugene Kooninand Kira Makarova]. Phylogenetic analysis based on the protein sequenceconservation of this set revealed five families of Cas9s, includingthree groups of large Cas9s (˜1400 amino acids) and two of small Cas9s(˜1100 amino acids) (see FIGS. 19 and 20A-F).

Further details of Cas9s and mutations of the Cas9 enzyme to convertinto a nickase or DNA binding protein and use of same with alteredfunctionality can be found in U.S. application Serial Nos 61/836,101 and61/835,936 (Broad Reference BI-2013/004E and BI-2013/004F respectively)incorporated herein by reference.

Example 6 Cas9 Orthologs

Applicants analyzed Cas9 orthologs to identify the relevant PAMsequences and the corresponding chimeric guide RNA. Having an expandedset of PAMs provides broader targeting across the genome and alsosignificantly increases the number of unique target sites and providespotential for identifying novel Cas9s with increased levels ofspecificity in the genome.

The specificity of Cas9 orthologs can be evaluated by testing theability of each Cas9 to tolerate mismatches between the guide RNA andits DNA target. For example, the specificity of SpCas9 has beencharacterized by testing the effect of mutations in the guide RNA oncleavage efficiency. Libraries of guide RNAs were made with single ormultiple mismatches between the guide sequence and the target DNA. Basedon these findings, target sites for SpCas9 can be selected based on thefollowing guidelines:

To maximize SpCas9 specificity for editing a particular gene, one shouldchoose a target site within the locus of interest such that potential‘off-target’ genomic sequences abide by the following four constraints:First and foremost, they should not be followed by a PAM with either5′-NGG or NAG sequences. Second, their global sequence similarity to thetarget sequence should be minimized. Third, a maximal number ofmismatches should lie within the PAM-proximal region of the off-targetsite. Finally, a maximal number of mismatches should be consecutive orspaced less than four bases apart.

Similar methods can be used to evaluate the specificity of other Cas9orthologs and to establish criteria for the selection of specific targetsites within the genomes of target species. As mentioned previouslyphylogenetic analysis based on the protein sequence conservation of thisset revealed five families of Cas9s, including three groups of largeCas9s (˜1400 amino acids) and two of small Cas9s (˜1100 amino acids)(see FIGS. 19 and 20A-F). Further details on Cas orthologs can be foundin U.S. application Serial Nos 61/836,101 and 61/835,936 (BroadReference BI-2013/004E and BI-2013/004F respectively) incorporatedherein by reference.

Example 7 Engineering of Plants (Micro-Algae) Using Cas9 to Target andManipulate Plant Genes

Methods of Delivering Cas9

Method 1: Applicants deliver Cas9 and guide RNA using a vector thatexpresses Cas9 under the control of a constitutive promoter such asHsp70A-Rbc S2 or Beta2-tubulin.

Method 2: Applicants deliver Cas9 and T7 polymerase using vectors thatexpresses Cas9 and T7 polymerase under the control of a constitutivepromoter such as Hsp70A-Rbc S2 or Beta2-tubulin. Guide RNA will bedelivered using a vector containing T7 promoter driving the guide RNA.

Method 3: Applicants deliver Cas9 mRNA and in vitro transcribed guideRNA to algae cells. RNA can be in vitro transcribed. Cas9 mRNA willconsist of the coding region for Cas9 as well as 3′UTR from Cop1 toensure stabilization of the Cas9 mRNA.

For Homologous recombination, Applicants provide an additional homologydirected repair template.

Sequence for a cassette driving the expression of Cas9 under the controlof beta-2 tubulin promoter, followed by the 3′ UTR of Cop1.

(SEQ ID NO: 19) TCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACATGTACCCATACGATGTTCCAGATTACGCTTCGCCGAAGAAAAAGCGCAAGGTCGAAGCGTCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTcAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTAcCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGGTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCGTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCGAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAGCCCCAAGAAGAAGAGAAAGGTGGAGGCCAGCTAAGGATCCGGCAAGACTGGCCCCGCTTGGCAACGCAACAGTGAGCCCCTCCCTAGTGTGTTTGGGGATGTGACTATGTATTCGTGTGTTGGCCAACGGGTCAACCCGAACAGATTGATACCCGCCTTGGCATTTCCTGTCAGAATGTAACGTCAGTTGATGGTACT

Sequence for a cassette driving the expression of T7 polymerase underthe control of beta-2 tubulin promoter, followed by the 3′ UTR of Cop1:

(SEQ ID NO: 20) TCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACatgcctaagaagaagaggaaggttaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctggctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagcacgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctcatcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggcaagcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccactctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgaggacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactcaacaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtctactcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatgctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtagcaagactctoagactatcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctccgatgttccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgtcctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgtacaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaatcaccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaagacatcgacatgaatcctgaggactcaccgcgtggaaacgtgagccgctgctgtgtaccgcaaggacaaggctcgcaagtacgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccatctggttccettacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgatatgaccaaaggactgcttacgaggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatccacggtgcaaactgtgcgggtgtcgacaaggttccgtccctgagcgcatcaagttcattgaggaaaaccacgagaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgactgatccttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgtttgacgggtcttgctaggcatccagcacttctccgcgatgaccgagatgaggtaggtggtcgcgcggttaacttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagcagacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaagtcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttcagtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagccagctattgattccggcaagggtagatgttcactcagccgaatcaggctgaggatacatggctaagctgatttgggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgctggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcctgatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagttccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgctcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacggaatcgaatcttttgcactgattcacgactccttcggtacgattccggctgacgctgcgaacctgttcaaagcagtgcgcgaaactatggttgacacatatgagtcagtgatgtactggctgatttctacgaccagttcgctgaccagttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatcttagagtcggacttcgcgttcgcgtaaGGATCCGGCAAGACTGGCCCCGCTTGGCAACGCAACAGTGAGCCCCTCCCTAGTGTGTTTGGGGATGTGACTATGTATTCGTGTGTTGGCCAACGGGTCAACCCGAACAGATTGATACCCGCCTTGGCATTTCCTGTCAGAATGTAACGTCAGTTGATGGTACT

Sequence of guide RNA driven by the T7 promoter (T7 promoter, Nsrepresent targeting sequence):

(SEQ ID NO: 21) gaaatTAATACGACTCACTATANNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttt

Gene Delivery:

Chlamydomonas reinhardtii strain CC-124 and CC-125 from theChlamydomonas Resource Center will be used for electroporation.Electroporation protocol follows standard recommended protocol from theGeneArt Chlamydomonas Engineering kit.

Also, Applicants generate a line of Chlamydomonas reinhardtii thatexpresses Cas9 constitutively. This can be done by usingpChlamyl(linearized using PvuI) and selecting for hygromycin resistantcolonies. Sequence for pChlamyl containing Cas9 is below. In this way toachieve gene knockout one simply needs to deliver RNA for the guideRNA.For homologous recombination Applicants deliver guideRNA as well as alinearized homologous recombination template.

pChlamyl-Cas9: (SEQ ID NO: 22)TGCGGTATTTCACACCGCATCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGTTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGTCGCTGAGGCTTGACATGATTGGTGCGTATGTTTGTATGAAGCTACAGGACTGATTTGGCGGGCTATGAGGGCGGGGGAAGCTCTGGAAGGGCCGCGATGGGGCGCGCGGCGTCCAGAAGGCGCCATACGGCCCGCTGGCGGCACCCATCCGGTATAAAAGCCCGCGACCCCGAACGGTGACCTCCACTTTCAGCGACAAACGAGCACTTATACATACGCGACTATTCTGCCGCTATACATAACCACTCAGCTAGCTTAAGATCCCATCAAGCTTGCATGCCGGGCGCGCCAGAAGGAGCGCAGCCAAACCAGGATGATGTTTGATGCTGGTATTTGAGCACTTGCAACCCTTATCCGGAAGCCCCCTGGCCCACAAAGGCTAGGCGCCAATGCAAGCAGTTCGCATGCAGCCCCTGGAGCGGTGCCCTCCTGATAAACCGGCCAGGGGGCCTATGTTCTTTACTTTTTTACAAGAGAAGTCACTCAACATCTTAAAATGGCCAGGTGAGTCGACGAGCAAGCCCGGCGGATCAGGCAGCGTGCTTGCAGATTTGACTTGCAACGCCCGCATTGTGTCGACGAAGGCTTTTGGCTCCTCTGTCGCTGTCTCAAGCAGCATCTAACCCTGCGTCGCCGTTTCCATTTGCAGGAGATTCGAGGTACCATGTACCCATACGATGTTCCAGATTACGCTTCGCCGAAGAAAAAGCGCAAGGTCGAAGCGTCCGACAAGAAGTACAGCATCGGCCTCTGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTICAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACCTACGACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATG CCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACAATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCCTACAAGAACCCTGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGCTGAGATCGTCTTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCFGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAGCCCCAAGAAGAAGAGAAAGGTGGAGGCCAGCTAACATATGATTCGAATGTCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGACCTCCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACATGACACAAGAATCCCTGTTACTTCTCGACCGTATTGATTCGGATGATTCCTACGCGAGCCTGCGGAACGACCAGGAATTCTGGGAGGTGAGTCGACGAGCAAGCCCGGCGGATCAGGCAGCGTGCTTGCAGATTTGACTTGCAACGCCCGCATTGTGTGCGAAGGCTTTTGGCTCCTCTGTCGCTGTCTCAAGCAGCATCTAACCCTGCGTCGCCGTTTCCATTTGCAGCCGCTGGCCCGCCGAGCCCTGGAGGAGCTCGGGCTGCCGGTGCCGCCGGTGCTGCGGGTGCCCGGCGAGAGCACCAACCCCGTACTGGTCGGCGAGCCCGGCCCGGTGATCAAGCTGTTCGGCGAGCACTGGTGCGGTCCGGAGAGCCTCGCGTCGGAGTCGGAGGCGTACGCGGTCCTGGCGGACGCCCCGGTGCCGGTGCCCCGCCTCCTCGGCCGCGGCGAGCTGCGGCCCGGCACCGGAGCCTGGCCGTGGCCCTACCTGGTGATGAGCCGGATGACCGGCACCACCTGGCGGTCCGCGATGGACGGCACGACCGACCGGAACGCGCTGCTCGCCCTGGCCCGCGAACTCGGCCGGGTGCTCGGCCGGCTGCACAGGGTGCCGCTGACCGGGAACACCGTGCTCACCCCCCATTCCGAGGTCTTCCCGGAACTGCTGCGGGAACGCCGCGCGGCGACCGTCGAGGACCACCGCGGGTGGGGCTACCTCTCGCCCCGGCTGCTGGACCGCCTGGAGGACTGGCTGCCGGACCTTGGACACGCTGCTGGCCGGCCGCGAACCCCGGTTCGTCCACGGCGACCTGCACGGGACCAACATCTTCGTGGACCTGGCCGCGACCGAGGTCACCGGGATCGTCGACTTCACCGACGTCTATGCGGGAGACTCCCGCTACAGCCTGGTGCAACTGCATCTCAACGCCTTCCGGGGCGACCGCGAGATCCTGGCCGCGCTGCTCGACGGGGCGCAGTGGAAGCGGACCGAGGACTTCGCCCGCGAACTGCTCGCCTTCACCTTCCTGCACGACTTCGAGGTGTTCGAGGAGACCCCGCTGGATCTCTCCGGCTTCACCGATCCGGAGGAACTGGCGCAGTTCCTCTGGGGGCCGCCGGACACCGCCCCCGGCGCCTGATAAGGATCCGGCAAGACTGGCCCCGCTTTGGCAACGCAACAGTGAGCCCCTCCCTAGTGTGTTTGGGGATGTGACTATGTATTCGTGTGTTGGCCAACGGGTCAACCCGAACAGATTGATACCCGCCTTGGCATTTCCTGTCAGAATGTAACGTCAGTTGATGGTAC T. 

For all modified Chlamydomonas reinhardtii cells, Applicants use PCR,SURVEYOR nuclease assay, and DNA sequencing to verify successfulmodification.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

REFERENCES

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What is claimed is:
 1. A method of altering expression of at least onegene product comprising introducing into a eukaryotic cell containingand expressing a DNA molecule having a target sequence and encoding thegene product an engineered, non-naturally occurring Clustered RegularlyInterspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas)system comprising one or more viral vectors comprising: a) a firstregulatory element operable in a eukaryotic cell operably linked to atleast one nucleotide sequence encoding a CRISPR-Cas system guide RNAthat hybridizes with the target sequence, and b) a second regulatoryelement operable in a eukaryotic cell operably linked to a nucleotidesequence encoding a Type-II Cas9 protein, wherein components (a) and (b)are located on same or different vectors of the system, whereby theguide RNA targets the target sequence and the Cas9 protein cleaves theDNA molecule, whereby expression of the at least one gene product isaltered; and, wherein the Cas9 protein and the guide RNA do notnaturally occur together.
 2. The method of claim 1, wherein theexpression of two or more gene products is altered.
 3. The method ofclaim 1, wherein the CRISPR-Cas system further comprises one or morenuclear localization signal(s) (NLS(s)).
 4. The method of claim 1,wherein the CRISPR-Cas system comprises a trans-activating cr (tracr)sequence.
 5. The method of claim 1, wherein the guide RNAs comprise aguide sequence fused to a tracr sequence.
 6. The method of claim 1,wherein the Cas9 protein is codon optimized for expression in theeukaryotic cell.
 7. The method of claim 1, wherein the eukaryotic cellis a mammalian or human cell.
 8. The method of claim 1, wherein theexpression of one or more gene products is increased.
 9. The method ofclaim 1, wherein the expression of one or more gene products isdecreased.
 10. The method of claim 1, wherein the one or more viralvectors are selected from the group consisting of retroviral,lentiviral, adenoviral, adeno-associated and herpes simplex viralvectors.
 11. A CRISPR-Cas system-mediated genome editing methodcomprising introducing into a eukaryotic cell containing and expressinga DNA molecule having a target sequence and encoding at least one geneproduct an engineered, non-naturally occurring CRISPR-Cas systemcomprising one or more vectors comprising: a) a first regulatory elementoperable in a eukaryotic cell operably linked to at least one nucleotidesequence encoding a CRISPR-Cas system guide RNA that hybridizes with thetarget sequence, and b) a second regulatory element operable in aeukaryotic cell operably linked to a nucleotide sequence encoding aType-II Cas9 protein, wherein components (a) and (b) are located on sameor different vectors of the system, whereby expression of the at leastone gene product is altered through the CRISPR-Cas system acting as tothe DNA molecule comprising the guide RNA directing sequence-specificbinding of the CRISPR-Cas system, whereby there is genome editing; and,wherein the Cas9 protein and the guide RNA do not naturally occurtogether.
 12. The method of claim 11, wherein the expression of two ormore gene products is altered.
 13. The method of claim 11, wherein theCRISPR-Cas system further comprises one or more NLS(s).
 14. The methodof claim 11, wherein the CRISPR-Cas system comprises a tracr sequence.15. The method of claim 11, wherein the Cas9 protein is codon optimizedfor expression in the eukaryotic cell.
 16. The method of claim 11,wherein the eukaryotic cell is a mammalian or human cell.
 17. The methodof claim 11, wherein the expression of one or more gene products isincreased.
 18. The method of claim 11, wherein the expression of one ormore gene products is decreased.
 19. An engineered, non-naturallyoccurring CRISPR-Cas system comprising one or more viral vectorscomprising: a) a first regulatory element operable in a eukaryotic celloperably linked to at least one nucleotide sequence encoding aCRISPR-Cas system guide RNA that hybridizes with a target sequence of aDNA molecule in a eukaryotic cell that contains the DNA molecule,wherein the DNA molecule encodes and the eukaryotic cell expresses atleast one gene product, b) a second regulatory element operable in aeukaryotic cell operably linked to a nucleotide sequence encoding aType-II Cas9 protein, wherein components (a) and (b) are located on sameor different vectors of the system, wherein the CRISPR-Cas systemcomprises a tracr sequence, whereby the guide RNA targets and hybridizeswith the target sequence and the Cas9 protein cleaves the DNA molecule,whereby expression of the at least one gene product is altered; and,wherein the Cas9 protein and the guide RNA do not naturally occurtogether.
 20. The system of claim 19, wherein the CRISPR-Cas systemfurther comprises one or more NLS(s).
 21. The system of claim 19,wherein the Cas9 protein is codon optimized for expression in theeukaryotic cell.
 22. The system of claim 19, wherein the eukaryotic cellis a mammalian or human cell.
 23. The system of claim 19, wherein theexpression of one or more gene products is increased.
 24. The system ofclaim 19, wherein the expression of one or more gene products isdecreased.
 25. The system of claim 19, wherein the one or more viralvectors are selected from the group consisting of retroviral,lentiviral, adenoviral, adeno-associated and herpes simplex viralvectors.
 26. An engineered, programmable, non-naturally occurring TypeII CRISPR-Cas system comprising a Cas9 protein and at least one guideRNA that targets and hybridizes to a target sequence of a DNA moleculein a eukaryotic cell, wherein the DNA molecule encodes and theeukaryotic cell expresses at least one gene product and the Cas9 proteincleaves the DNA molecule and wherein the CRISP R-Cas system comprises atracr sequence, whereby expression of the at least one gene product isaltered; and, wherein the Cas9 protein and the guide RNA do notnaturally occur together.
 27. The CRISPR-Cas system of claim 26, whereinthe CRISPR-Cas system further comprises one or more NLS(s).
 28. TheCRISPR-Cas system of claim 26, wherein the Cas9 protein is codonoptimized for expression in the eukaryotic cell.
 29. The CRISPR-Cassystem of claim 26, wherein the eukaryotic cell is a mammalian or humancell.